Gas-phase purification for accurate isobaric tag-based quantification

ABSTRACT

Described herein are mass spectrometry systems and methods which improve the accuracy of isobaric tag-based quantification by alleviating the pervasive problem of precursor interference and co-isolation of impurities through gas-phase purification. During the gas-phase purification, the mass-to-charge ratios of precursor ions within at least a selected range are selectively changed allowing ions having similar unmodified mass-to-charge ratios to be separated before further isolation, fragmentation or analysis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority under 35 U.S.C.119(e) to U.S. Provisional Application 61/471,461 filed on Apr. 4, 2011entitled “Gas-Phase Purification For Accurate Isobaric Tag-BasedQuantification”, which is hereby incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM080148 andGM081629 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

The ability to identify proteins and determine their chemical structureshas become central to the life sciences. The amino acid sequence ofproteins provides a link between proteins and their coding genes via thegenetic code, and, in principle, a link between cell physiology andgenetics. The identification of proteins provides a window into complexcellular regulatory networks.

Mass spectrometry (MS), including but not limited to triple quadropoleand ion trap mass spectrometers, is among the most widely used platformsfor molecular analysis and identification—spanning natural products,pharmaceuticals and biologics. Most mass spectrometer-based experimentsbegin with the isolation of a group of compounds from a set of samplesthrough some sort of extraction technique, such as extraction ofproteins from tissues, cell lysates or fluids followed by proteolyticdigestion of those proteins into peptides (i.e., bottom-up proteomics).Frequently, but not necessarily, mass spectrometers are coupled withsome form of separation, such as electrophoretic or chromatographicseparation systems. Over the course of just a few hours, mass spectralinstruments can autonomously interrogate tens of thousands of molecularspecies via tandem mass spectrometry (MS/MS).

Quantitative analysis in chemistry is the determination of the absoluteor relative abundance of one, several, or all particular substance(s)present in a sample. For biological samples, quantitative analysisperformed via mass spectrometry can determine the relative abundance ofpeptides and proteins. The accepted methodology for performing massspectrometric quantitation is accomplished using a mass spectrometercapable of MS/MS fragmentation (i.e., triple quadropole or ion trapspectrometers). The quantitation process can involve isobaric tagging ofpeptide precursors, which when combined with post-acquisition software,provides the relative abundance of peptides. However, when a peptideprecursor is selected for tandem mass spectrometry, there are ofteninterfering species with similar mass-to-charge ratios that areco-isolated and subjected to activation. These species are often otherisobarically tagged peptides with different relative quantitation, whichtherefore disturb the quantitative measurement of the peptide ofinterest.

As a result, protein identification technologies have rapidly maturedsuch that constructing catalogs of the thousands of proteins comprisedby a cell using mass spectrometry is now relatively straightforward [deGodoy, L. M. F. et al. Nature 455, 1251-1255 (2008); Swaney, D. L.,Wenger, C. D. & Coon, J. J. J. Proteome Res. 9, 1323-1329 (2010)];however, knowing how the abundance of these molecules change undervarious circumstances is not [Ong, S. E. & Mann, M. Nat. Chem. Biol. 1,252-262 (2005)]. Stable isotope labeling by amino acids in cell culture(SILAC) provides a means to make binary or ternary comparisons [Jiang,H. & English, A. M. J. Proteome Res. 1, 345-350 (2002); Ong, S. E. etal. Mol. Cell. Proteomics 1, 376-386 (2002)]. By interlacing these two-or three-way experiments, higher-order comparisons can be obtained[Olsen, J. V. et al. Sci. Signal. 3, ra3 (2010)]. Such large-scalemultiplexed experiments are invaluable, as they (1) allow measurement oftime-course experiments, (2) permit collection of biological replicates,and (3) enable direct comparison of transcriptomic and proteomic data.

Constructing this type of multi-faceted proteomics study, however, is anarduous undertaking and has only been accomplished in a handful ofexperiments by an even smaller group of researchers. The firstimpediment is the requirement to grow multiple groups of cells withvarious labels. This step is actually less limiting than the secondmajor obstacle: each binary or ternary set must be analyzed separately.When combined with the need for extensive pre-MS fractionation andtechnical replicates, a large-scale experiment via SILAC demands threeto six months of constant instrument usage.

Isobaric tagging [Thompson, A. et al. Anal. Chem. 75, 1895-1904 (2003);Ross, P. L. et al. Mol. Cell. Proteomics 3, 1154-1169 (2004)], is anelegant solution to this problem, allowing relative quantification of upto eight proteomes simultaneously [Choe, L. et al. Proteomics 7,3651-3660 (2007); Dayon, L. et al. Anal. Chem. 80, 2921-2931 (2008)].Further, it is compatible with mammalian tissues and biofluids, unlikemetabolic approaches. Despite its potential to enable fast, multiplexedquantitative proteomics, isobaric tagging has not been widely embracedfor large-scale studies [Lu, R. et al. Nature 462, 358-U126(2009)]—chiefly because of precursor interference. This problem does notexist for SILAC because abundance measurements are performed withhigh-resolution MS¹ analysis in tandem mass spectrometry. Even for verycomplex samples having tens or hundreds of co-eluting peptides,high-resolving power mass analyzers can easily distinguish the targetfrom neighboring peaks less than 0.01 Th away.

In the isobaric approach, however, the target peptide is isolated atmuch lower resolution, typically 1-3 Th, and dissociated to producereporter tags. Therefore, the quantitative signal in the reporter regionis compiled from every species in the isolation window [Ow, S. Y. et al.J. Proteome Res. 8, 5347-5355 (2009)]. For highly complex mixtures, likethose analyzed in large-scale experiments, co-isolation of multiplespecies is the rule, not the exception (vide infra). This problem erodesquantitative accuracy, as measured ratios tend to be compressed towardthe median ratio of 1:1, and thus has restricted isobaric tagging toapplications with lower sample complexity.

Isobaric labeling, such as iTRAQ and other types if isobaric taggingreagents, is an important quantitative method as it allows formultiplexing and is directly applicable to clinical samples. Asignificant source of error, however, occurs when another elutingpeptide ion has a m/z value that is very near that of the selectedprecursor (˜50%, in many experiments). The result is the isolation ofboth species, which are consequently co-dissociated, to produce acomposite MS/MS spectrum. The resulting reporter ion ratios do notaccurately reflect the relative abundances of either peptide; limitingboth the precision and dynamic range of quantitation, as the medianpeptide ratio is close to 1:1.

The increasing popularity of iTRAQ for quantitative proteomicsapplications has spurred increased efforts to evaluate its relevance,accuracy, and precision for biological interpretation. Recently, someresearchers have begun to assess the accuracy and precision of iTRAQquantification as well as drawbacks which hinder the applicability andattainable dynamic range of iTRAQ. Some results suggest that crosstalkbetween interfering factors can result in underestimations. [Ow et al.,“iTRAQ Underestimation in Simple and Complex Mixtures: ‘The Good, theBad and the Ugly”, Journal of Proteome Research, web publication Sep.16, 2009]. It is clear that there is tantalizing potential for iTRAQ andother protein labeling methods to provide accurate quantificationspanning several orders of magnitude. This potential can be limited,however, by several factors. First, for example, the existence ofisotopic impurities often requires correction of mass spectral data toprovide accurate quantitation which currently requires the availabilityof accurate isotopic factors. Second, the interference of mixed MS/MScontribution occurring during precursor selection is a problem that iscurrently very difficult to minimize.

What is needed is a method of improving the accuracy of massspectrometry analysis and quantification of samples, particularlysamples labeled with isobaric tags.

SUMMARY OF THE INVENTION

The present invention provides systems and methods which utilizegas-phase purification to improve mass spectrometry analysis andquantification. During gas-phase purification, precursor ions aregenerated from an analyte during a first ionization step, such as theMS¹ stage in tandem mass spectrometry. The mass-to-charge ratios of atleast a selected range of these precursor ions are manipulated ormodified allowing ions having similar unmodified mass-to-charge ratiosto be separated before further isolation, fragmentation and/or analysis.Optionally, mass spectrometry data generated from changing themass-to-charge ratios of the precursor ions is used in conjunction withdata generated from analysis of the unmodified precursor ions to provideadditional information and identification. In particular, methods andsystems described herein improve the accuracy of isobaric tag-basedquantification by alleviating the problem of precursor interference andco-isolation of impurities.

In one embodiment, the present invention provides a method of analyzingan analyte using mass spectrometry, where the method comprises:

-   -   (a) providing an isobarically labeled analyte;    -   (b) generating a first distribution of precursor ions from the        isobarically labeled analyte;    -   (c) identifying a range of mass-to-charge ratios of the first        distribution of precursor ions;    -   (d) selectively changing the mass-to-charge ratios of precursor        ions in the identified range of mass-to-charge ratios, thereby        generating a distribution of mass-to-charge-manipulated        precursor ions;    -   (e) isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions,        thereby generating isolated mass-to-charge-manipulated precursor        ions;    -   (f) fragmenting ions corresponding to the isolated        mass-to-charge-manipulated precursor ions, thereby generating        first product ions; and    -   (g) measuring the mass-to-charge ratios of the first product        ions, thereby generating first product ion mass spectrometry        data;        thereby analyzing the analyte using mass spectrometry. In an        embodiment, the method further comprises providing an analyte,        such as a protein or peptide analyte, and isobarically labeling        the analyte so as to generate an isobarically labeled analyte.

In an embodiment, the range of mass-to-charge ratios of the firstdistribution of precursor ions in step (c) is referred to as the firstisolation window and can vary in width. Increased purification will beachieved the narrower this window is around the target mass-to-chargeratio value; however, narrowing the isolation window will also reducethe amount of desired precursor ions that will have their charge or massmanipulated and analyzed leading to less identification. In oneembodiment, the identified range of mass-to-charge ratios of the firstdistribution of precursor ions in step (c) has a width of 3 m/z units orless. In one embodiment, this first isolation window has a width of 0.1to 10 m/z units, a width of 0.1 to 5 m/z units, a width of 0.5 to 4 m/zunits, a width of 1 to 3 m/z units, a width of 1 to 2 m/z units, or awidth of 2.5 to 3.5 m/z units centered on the post-manipulation targetmass-to-charge ratio value. Wider widths up to about 100 or moremass-to-charge units can be applied. Decreasing the width to a verynarrow isolation range (up to about 0.1 mass-to-charge units) providesincreased purification

In an embodiment, the method further comprises:

-   -   (h) generating a second distribution of precursor ions from the        isobarically labeled analyte;    -   (i) identifying a range of mass-to-charge ratios of the second        distribution of precursor ions;    -   (j) fragmenting ions corresponding to the range of        mass-to-charge ratios of the second distribution of precursor        ions, thereby generating second product ions; and    -   (k) measuring the mass-to-charge ratios of the second product        ions, thereby generating second product ion mass spectrometry        data.

In an embodiment, the step of:

-   -   (d) selectively changing the mass-to-charge ratios of precursor        ions in the identified range of mass-to-charge ratios, thereby        generating a distribution of mass-to-charge-manipulated        precursor ions;        can be performed so that the resulting products have a        predictable mass-to-charge ratio change. The mass-to-charge        ratio of the precursor ions can be changed by manipulating the        mass of the precursor ions within the identified range, or by        manipulating the charge of the precursor ions. Charge-transfer        reactions, for example, remove charge so that the new        mass-to-charge ratio value of the target can be calculated,        without need for a separate analysis, so that the step of:    -   (e) isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions,        thereby generating isolated mass-to-charge-manipulated precursor        ions;        can be applied immediately following the manipulation step. This        second isolation window can vary in width. Increased        purification will be achieved the narrower this window is around        the post-manipulation target mass-to-charge ratio value;        however, narrowing the isolation window will also reduce the        amount of desired charge-manipulated precursor ions that will be        fragmented and analyzed leading to less identification. In one        embodiment, this second isolation window has a width of 0.1 to        10 m/z units, a width of 0.1 to 5 m/z units, a width of 0.5 to 4        m/z units, a width of 1 to 3 m/z units, a width of 1 to 2 m/z        units, or a width of 2.5 to 3.5 m/z units centered on the        post-manipulation target mass-to-charge ratio value. Wider        widths up to about 100 or more mass-to-charge units can be        applied. Decreasing the width to a very narrow isolation range        (up to about 0.1 mass-to-charge units) provides increased        purification.

In an embodiment, the range of mass-to-charge ratios of the seconddistribution of precursor ions is identified so as not to includemass-to-charge ratios corresponding to reporter ions of the isobaricallylabeled analyte. In an embodiment, the identified range ofmass-to-charge ratios of the second distribution of precursor ions doesnot include ions having a mass-to-charge ratio less than 400 m/z units.In an embodiment, the identified range of mass-to-charge ratios of thesecond distribution of precursor ions does not include ions having amass-to-charge ratio less than 200 m/z units. In an embodiment, theidentified range of mass-to-charge ratios of the second distribution ofprecursor ions does not include ions having a mass-to-charge ratio lessthan 175 m/z units. In an embodiment, the identified range ofmass-to-charge ratios of the second distribution of precursor ions doesnot include ions having a mass-to-charge ratio less than 150 m/z units.

In an embodiment, the identified range of mass-to-charge ratios of thefirst distribution of precursor ions and the identified range ofmass-to-charge ratios of the first distribution of precursor ions arenot the same. In another embodiment, the identified ranges ofmass-to-charge ratios of the first and second distributions of precursorions are the same. In an embodiment, the identified ranges ofmass-to-charge ratios of the first and second distributions,independently from one another, have a width of 100 m/z units or less,10 m/z units or less, 5 m/z units or less, 3 m/z units or less, 2 m/zunits or less, or 1 m/z unit or less. In an embodiment, the identifiedranges of mass-to-charge ratios of the first and second distributions,independently from one another, have a width of 0.1 to 10 m/z units, awidth of 0.1 to 5 m/z units, a width of 0.5 to 4 m/z units, a width of 1to 3 m/z units, a width of 1 to 2 m/z units, or a width of 2.5 to 3.5m/z units centered on the post-manipulation target mass-to-charge ratiovalue.

In an embodiment, this method further comprises storing the firstproduct ions and the second product ions concurrently in a single ionstorage device before measuring the mass-to-charge ratios of the firstproduct ions and the second product ions. In an alternate embodiment,the method further comprises storing the first product ions and thesecond product ions concurrently in separate ion storage devices beforemeasuring the mass-to-charge ratios of the first product ions and secondproduct ions.

In another embodiment, the method further comprises storing the firstproduct ions and the second product ions sequentially in a single ionstorage device before measuring the mass-to-charge ratios of the firstproduct ions and second product ions.

In an embodiment, the steps of:

-   -   (g) measuring the mass-to-charge ratios of the first product        ions, thereby generating product ion mass spectrometry data; and    -   (k) measuring the mass-to-charge ratios of the second product        ions, thereby generating product ion mass spectrometry data;        are performed concurrently using a single mass analyzer.

Alternatively, the steps of:

-   -   (g) measuring the mass-to-charge ratios of the first product        ions, thereby generating product ion mass spectrometry data; and    -   (k) measuring the mass-to-charge ratios of the second product        ions, thereby generating product ion mass spectrometry data;        are performed sequentially using a single mass analyzer.

In an embodiment, the steps of:

-   -   (g) measuring the mass-to-charge ratios of the first product        ions, thereby generating product ion mass spectrometry data; and    -   (k) measuring the mass-to-charge ratios of the second product        ions, thereby generating product ion mass spectrometry data;        are performed concurrently or non-concurrently using separate        mass analyzers.

In an embodiment, the first distribution of precursor ions is subject tocollisional dissociation to selectively change the mass-to-charge ratiosof precursor ions in the identified range of mass-to-charge ratios,thereby generating a distribution of mass-to-charge-manipulatedprecursor ions. In an embodiment, for example, the method of theinvention further comprises the step of collisionally dissociatingprecursor ions in the identified range of mass-to-charge ratios, therebygenerating the distribution of mass-to-charge-manipulated precursorions. In an embodiment, the first distribution of precursor ions issubject to photodissociation to selectively change the mass-to-chargeratios of precursor ions in the identified range of mass-to-chargeratios, thereby generating a distribution of mass-to-charge-manipulatedprecursor ions. In an embodiment, for example, the method of theinvention further comprises the step of photodissociating precursor ionsin the identified range of mass-to-charge ratios, thereby generating thedistribution of mass-to-charge-manipulated precursor ions.Alternatively, in an embodiment, the first distribution of precursorions is not subject to collisional dissociation to selectively changethe mass-to-charge ratios of precursor ions in the identified range ofmass-to-charge ratios and/or the first distribution of precursor ions isnot subject to photodissociation to selectively change themass-to-charge ratios of precursor ions in the identified range ofmass-to-charge ratios, thereby generating a distribution ofmass-to-charge-manipulated precursor ions.

In an embodiment, the first distribution of precursor ions is subject toa reaction with a species to selectively change the mass-to-chargeratios of precursor ions in the identified range of mass-to-chargeratios, thereby generating a distribution of mass-to-charge-manipulatedprecursor ions. In an embodiment, for example, the method of theinvention further comprises the step of reacting the precursor ions inthe identified range of mass-to-charge ratios with a species (such as acharge manipulation reactant) so as to generate the distribution ofmass-to-charge-manipulated precursor ions. In an embodiment, thereaction with a species to selectively change the mass-to-charge ratiosof the range of mass-to-charge ratios comprises an ion-moleculereaction. In an embodiment, the reaction with a species to selectivelychange the mass-to-charge ratios of the range of mass-to-charge ratioscomprises an ion-ion reaction. In an embodiment, the reaction with aspecies to selectively change the mass-to-charge ratios of the range ofmass-to-charge ratios comprises an ion-electron reaction.

Many different species are useful in a reaction with a species toselectively change the mass-to-charge ratios of precursor ions in theidentified range of mass-to-charge ratios, thereby generating adistribution of mass-to-charge-manipulated precursor ions. In anembodiment, for example, singly or multiply charged anions of peptides,proteins, oligonucleotides, biological molecules, polymers, ordendrimers are useful in a reaction with a species to selectively changethe mass-to-charge ratios of precursor ions in the identified range ofmass-to-charge ratios, thereby generating a distribution ofmass-to-charge-manipulated precursor ions. In another embodiment anychemical species with a suitable mass (i.e. >20 Da) is useful in areaction with a species to selectively change the mass-to-charge ratiosof precursor ions in the identified range of mass-to-charge ratios,thereby generating a distribution of mass-to-charge-manipulatedprecursor ions.

In an embodiment, the reaction with a species to selectively change themass-to-charge ratios of the range of mass-to-charge ratios comprises aproton-transfer reaction. In an embodiment, the proton transfer reactioncomprises reaction of the first distribution of precursor ions withfluoranthene, perfluoro-1,3-dimethyl-cyclohexane, 2,6-dichlorophenol,2,3,4,6-tetrachlorophenol, or 1,4-naphthoquinone.

In an embodiment, the reaction with a species to selectively change themass-to-charge ratios of the range of mass-to-charge ratios comprises acharge-transfer reaction. In an embodiment, the reaction with a speciesto selectively change the mass-to-charge ratios of the range ofmass-to-charge ratios comprises a electron-transfer reaction.

In an embodiment, the reaction with a species to selectively change themass-to-charge ratios of the range of mass-to-charge ratios is areaction that changes the mass of the precursor ions by a known amount.In one embodiment, the mass of each precursor ion is modified by 200atomic mass units or less, 100 atomic mass units or less, 75 amu atomicmass units or less, 50 atomic mass units or less, 25 atomic mass unitsor less, or 10 atomic mass units or less.

In an embodiment, the reaction with a species to selectively change themass-to-charge ratios of the range of mass-to-charge ratios is areaction that changes the charge state of the precursor ions. Thischange in charge state is predictable in that the charge state will beincreased or decreased by a known amount. In one embodiment, theprecursor ions in the identified range of mass-to-charge ratios have apositive charge state, and selectively changing the mass-to-chargeratios of the precursor ions in the identified range comprisesincreasing or decreasing the positive charge state of each of theprecursor ions. In one embodiment, the positive charge state of theprecursor ion is increased by 1, by 2, or by 3. In a further embodiment,the positive charge state of the precursor ion is decreased by 1, by 2,or by 3. In a further embodiment, the positive charge state of theprecursor ion is decreased by one.

In an embodiment, the step of:

-   -   (e) isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions,        thereby generating isolated mass-to-charge-manipulated precursor        ions;        comprises isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions        corresponding to 100 m/z units or less.

In an embodiment, the step of:

-   -   (e) isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions,        thereby generating isolated mass-to-charge-manipulated precursor        ions;        comprises isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions        corresponding to 3 m/z units or less.

In an embodiment, the step of:

-   -   (e) isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions,        thereby generating isolated mass-to-charge-manipulated precursor        ions;    -   comprises isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions        corresponding to 1 m/z units or less.

In an embodiment, the step of:

-   -   (e) isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions,        thereby generating isolated mass-to-charge-manipulated precursor        ions;        comprises isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions        corresponding to 0.2 m/z units or less.

In an embodiment, the step of:

-   -   (e) isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions,        thereby generating isolated mass-to-charge-manipulated precursor        ions;        comprises isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions        corresponding to 0.1 m/z units or less.

In an embodiment, the method further comprises analyzing themass-to-charge ratios of at least a portion of the first distribution ofprecursor ions, thereby generating first precursor ion mass spectrometrydata corresponding to the first distribution of precursor ions.

In an embodiment, the method further comprises analyzing themass-to-charge ratios of at least a portion of themass-to-charge-manipulated precursor ions, thereby generatingmass-to-charge-manipulated precursor ion mass spectrometry datacorresponding to the distribution of mass-to-charge-manipulatedprecursor ions.

In an embodiment, the method further comprises analyzing themass-to-charge ratios of at least a portion of the second distributionof precursor ions, thereby generating second precursor ion massspectrometry data corresponding to the second distribution of precursorions.

In an embodiment, the step of:

-   -   (f) fragmenting ions corresponding to the isolated        mass-to-charge-manipulated precursor ions, thereby generating        first product ions;        comprises fragmenting the isolated mass-to-charge-manipulated        precursor ions by beam-type collisionally activated        dissociation, ultraviolet photo-dissociation, infrared        photodissociation, electron transfer dissociation, electron        capture dissociation, surface induced dissociation, or resonant        excitation collisionally activated dissociation.

In an embodiment, the step of:

-   -   (j) fragmenting ions corresponding to the second distribution of        precursor ions, thereby generating second product ions;        comprises fragmenting the ions corresponding to the second        distribution of precursor ions by beam-type collisionally        activated dissociation, ultraviolet photodissociation, infrared        photodissociation, electron transfer dissociation, electron        capture dissociation, surface induced dissociation, or resonant        excitation collisionally activated dissociation.

In an embodiment, the reaction conditions and ion optics are adjusted tomaximize the amount of first product ions generated. In an embodiment,the reaction conditions and ion optics are adjusted to maximize theamount of second product ions generated.

In an embodiment, at least a portion of the first product ion massspectrometry data corresponds to a reporter tag of the isobaricallylabeled analyte. In an embodiment, at least a portion of the secondproduct ion mass spectrometry data corresponds to a sequence tag of theisobarically labeled analyte.

In an embodiment, the first distribution of precursor ions is generatedby an electrospray ionization source or a MALDI source. In anembodiment, the second distribution of precursor ions is generated by anelectrospray ionization source or a MALDI source.

In an embodiment, the isobarically labeled analyte comprises proteins orpeptides. In an embodiment, the isobarically labeled analyte comprisesphosphorylated proteins or peptides. In an embodiment, the isobaricallylabeled analyte comprises co-translationally modified proteins orpeptides. In an embodiment, the isobarically labeled analyte comprisespost-translationally modified proteins or peptides. In an embodiment,the isobarically labeled analyte comprises small molecules,pharmaceutical compounds, oligonucleotides, or sugars. In an embodiment,the isobarically labeled analyte comprises isobarically labeled proteinsor peptides. In an embodiment, the isobarically labeled analyte isfractionated prior to generating the first distribution of precursorions from the analyte. In an embodiment, the isobarically labeledanalyte is fractionated prior to generating the second distribution ofprecursor ions from the analyte.

In an embodiment, the method is implemented in a tandem massspectrometer instrument, a multistage mass spectrometer instrument, or ahybrid mass spectrometer instrument.

In an embodiment, the isobarically labeled analyte comprises proteins orpeptides and the isobarically labeled analyte is analyzed to quantifythe amount of proteins or peptides in the analyte. In an embodiment,wherein the isobarically labeled analyte comprises one or more proteins.

In an embodiment the method further comprises digesting the one or moreproteins. In an embodiment the method further comprises identifyingpeptides corresponding to the one or more proteins. In an embodiment themethod further comprises determining amounts of the one or moreproteins. In an embodiment the method further comprises determining acomposition of the one or more proteins. In an embodiment the methodfurther comprises determining a post translational modification of theone or more proteins. In an embodiment, the one or more proteins areindicative of a disease state.

Another embodiment provides a method able to be used with stand-aloneion traps (termed “low-resolution QuantMode” or “low-res QuantMode”)which drastically improves the quantitative accuracy and dynamic rangeachievable on low-resolution MS instrumentation for isobaric tag-basedquantitative analyses of complex samples. The problem of precursorinterference is addressed through the use of gas-phase purification andthe utilization of carefully designed segmented scan sequences whichenables the generation of both optimal reporter ions and optimalsequencing ions for analysis, while minimizing unnecessary drains on theduty cycle of the mass spectrometer.

In one embodiment, the present invention provides a method of analyzingan analyte using mass spectrometry, where the method comprises:

-   -   (a) providing an isobarically labeled analyte;    -   (b) generating a first distribution of precursor ions from the        isobarically labeled analyte;    -   (c) identifying a range of mass-to-charge ratios of the first        distribution of precursor ions;    -   (d) scanning the precursor ions in the identified range of        mass-to-charge ratios to determine the charge state of the        identified precursor ions;    -   (e) reacting the precursor ions in the identified range of        mass-to-charge ratios with a species to selectively change the        mass-to-charge ratios of precursor ions in the identified range        of mass-to-charge ratios, wherein the reaction with a species to        selectively change the mass-to-charge ratios of the range of        mass-to-charge ratios comprises a proton-transfer reaction        thereby generating a distribution of mass-to-charge-manipulated        precursor ions having a decreased positive charge state;    -   (f) isolating a range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions,        thereby generating isolated mass-to-charge-manipulated precursor        ions;    -   (g) fragmenting ions corresponding to the isolated        mass-to-charge-manipulated precursor ions, thereby generating        first product ions; and    -   (h) measuring the mass-to-charge ratios of the first product        ions, thereby generating first product ion mass spectrometry        data,        thereby analyzing the analyte using mass spectrometry. If the        determined charge state of the identified precursor ions is +2        or greater, then isolating the range of mass-to-charge ratios of        the distribution of mass-to-charge-manipulated precursor ions is        based on the determined charge state of a desired precursor ion.

However, if the determined charge state of the identified precursor ionsis unknown or is less than +2, then isolating the range ofmass-to-charge ratios of the distribution of mass-to-charge-manipulatedprecursor ions is based on a desired charge state of +3. Additionally asecond quantification scan is performed and the method further comprisesthe steps of:

-   -   (i) isolating a second range of mass-to-charge ratios of the        distribution of mass-to-charge-manipulated precursor ions,        thereby generating second isolated mass-to-charge-manipulated        precursor ions;    -   (j) fragmenting ions corresponding to the second isolated        mass-to-charge-manipulated precursor ions, thereby generating        second product ions; and    -   (k) measuring the mass-to-charge ratios of the second product        ions, thereby generating second product ion mass spectrometry        data.

The invention also provides systems for performing mass spectrometry. Inan embodiment, a mass spectrometer system for analyzing an isobaricallylabeled analyte is provided, the system comprising:

-   -   an ion source for generating ions from the isobarically labeled        analyte;    -   first ion separation optics in communication with the ion source        for separating ions according to their mass-to-charge ratios;    -   ion reaction optics in communication with the first ion        separation optics for generating mass-to-charge-manipulated        ions;    -   ion fragmentation optics in communication with the first ion        separation optics for generating product ions;    -   second ion separation optics in communication with the ion        fragmentation optics for separating ions according to their        mass-to-charge ratios;    -   a first ion detector in communication with the second ion        separation optics for detecting ions separated according to        their mass-to-charge ratios;    -   a controller operably connected to the first and second ion        separation optics, the ion reaction optics, the first ion        detector, and the ion fragmentation optics;        wherein the controller controls the ion optics and detector so        as to:    -   (a) generate a first distribution of precursor ions from the        isobarically labeled analyte;    -   (b) identify a range of mass-to-charge ratios of the first        distribution of precursor ions;    -   (c) selectively change the mass-to-charge ratios of precursor        ions in the identified range of mass-to-charge ratios, thereby        generating a distribution of mass-to-charge-manipulated        precursor ions;    -   (d) isolate a range of mass-to-charge ratios of the distribution        of mass-to-charge-manipulated precursor ions, thereby generating        isolated mass-to-charge-manipulated precursor ions;    -   (e) fragment ions corresponding to the isolated        mass-to-charge-manipulated precursor ions, thereby generating        first product ions;    -   (f) measure the mass-to-charge ratios of the first product ions,        thereby generating first product ion mass spectrometry data; and    -   (g) analyze the first product ion mass spectrometry data. As        used throughout the present description, the term “ion optics”        is intended to be inclusive of ion optic components of a mass        spectrometer system, including, for example, one or more ion        separation optics, ion reaction optics, ion fragmentation optics        and combinations thereof. As used throughout the present        description, the term “detector” is intended to be inclusive of        detector components of a mass spectrometer system, including,        for example, one or more ion detectors.

In an embodiment, the controller further controls the ion optics anddetector so as to:

-   -   (h) generate a second distribution of precursor ions from the        isobarically labeled analyte;    -   (i) identify a range of mass-to-charge ratios of the second        distribution of precursor ions;    -   (j) fragment ions corresponding to the range of mass-to-charge        ratios of the second distribution of precursor ions, thereby        generating second product ions;    -   (k) measure the mass-to-charge ratios of the second product        ions, thereby generating second product ion mass spectrometry        data; and    -   (l) analyze the second product ion mass spectrometry data.

In an embodiment, the system further comprises a first ion storagedevice in communication with the ion fragmentation optics and second ionseparation optics and operably connected to the controller.

In an embodiment, the controller further controls the first ion storagedevice to store the first product ions and the second product ionsconcurrently in the ion storage device before measuring themass-to-charge ratios of the first product ions and the second productions.

In an embodiment, the controller further controls the first ion storagedevice to store the first product ions and the second product ionssequentially in the ion storage device before measuring themass-to-charge ratios of the first product ions and the second productions.

In an embodiment, the system further comprises a second ion storagedevice in communication with the ion fragmentation optics and second ionseparation optics and operably connected to the controller. In anembodiment, the controller further controls the first ion storage deviceand second ion storage device to store the first product ions in thefirst ion storage device and the second product ions in the second ionstorage device concurrently before measuring the mass-to-charge ratiosof the first product ions and the second product ions. However, itshould be noted that the controller can also control the first andsecond storage devices to the store the first and second product ionsnon-currently.

In an embodiment, the controller controls the ion optics and detector soas to:

-   -   (f) measure the mass-to-charge ratios of the first product ions,        thereby generating product ion mass spectrometry data; and    -   (k) measure the mass-to-charge ratios of the second product        ions, thereby generating product ion mass spectrometry data;        concurrently with the second ion separation optics and first        detector.

In an embodiment, the controller controls the ion optics and detector soas to:

-   -   (f) measure the mass-to-charge ratios of the first product ions,        thereby generating product ion mass spectrometry data; and    -   (k) measure the mass-to-charge ratios of the second product        ions, thereby generating product ion mass spectrometry data;        sequentially with the second ion separation optics and first        detector.

In an embodiment, the system further comprises:

-   -   third ion separation optics in communication with the ion        fragmentation optics, for separating ions according to their        mass-to-charge ratios;    -   a third detector in communication with the third ion separation        optics, for detecting ions separated according to their        mass-to-charge ratios;        wherein the controller controls the ion optics and detectors so        as to:    -   (f) measure the mass-to-charge ratios of the first product ions,        thereby generating product ion mass spectrometry data; and    -   (k) measure the mass-to-charge ratios of the second product        ions, thereby generating product ion mass spectrometry data;        concurrently with the second ion separation optics and first        detector and the third ion separation optics and third detector.

In an embodiment, the controller further controls the ion optics anddetectors to: subject the first distribution of precursor ions tocollisional dissociation to selectively change the mass-to-charge ratiosof precursor ions in the identified range of mass-to-charge ratios,thereby generating a distribution of mass-to-charge-manipulatedprecursor ions.

In an embodiment, the controller further controls the ion optics anddetectors to: subject the first distribution of precursor ions tophotodissociation to selectively change the mass-to-charge ratios ofprecursor ions in the identified range of mass-to-charge ratios, therebygenerating a distribution of mass-to-charge-manipulated precursor ions.

In an embodiment, the controller does not further control the ion opticsand detectors to: subject the first distribution of precursor ions tocollisional dissociation to selectively change the mass-to-charge ratiosof precursor ions in the identified range of mass-to-charge ratios,thereby generating a distribution of mass-to-charge-manipulatedprecursor ions.

In an embodiment, the controller does not further control the ion opticsand detectors to: subject the first distribution of precursor ions tophotodissociation to selectively change the mass-to-charge ratios ofprecursor ions in the identified range of mass-to-charge ratios, therebygenerating a distribution of mass-to-charge-manipulated precursor ions.

In an embodiment, the controller further controls the ion optics anddetectors to: subject the first distribution of precursor ions toreaction with a species to selectively change the mass-to-charge ratiosof precursor ions in the identified range of mass-to-charge ratios,thereby generating a distribution of mass-to-charge-manipulatedprecursor ions. In an embodiment, reaction with a species to selectivelychange the mass-to-charge ratios of the range of mass-to-charge ratioscomprises an ion-molecule reaction. In an embodiment, reaction with aspecies to selectively change the mass-to-charge ratios of the range ofmass-to-charge ratios comprises an ion-ion reaction. In an embodiment,reaction with a species to selectively change the mass-to-charge ratiosof the range of mass-to-charge ratios comprises an ion-electronreaction. In an embodiment, reaction with a species to selectivelychange the mass-to-charge ratios of the range of mass-to-charge ratioscomprises a proton-transfer reaction. In an embodiment, the protontransfer reaction comprises reaction of the first distribution ofprecursor ions with fluoranthene, perfluoro-1,3-dimethyl-cyclohexane,2,6-dichlorophenol, 2,3,4,6-tetrachlorophenol, or 1,4-naphthoquinone.

In an embodiment, reaction with a species to selectively change themass-to-charge ratios of the range of mass-to-charge ratios comprises acharge-transfer reaction. In an embodiment, reaction with a species toselectively change the mass-to-charge ratios of the range ofmass-to-charge ratios comprises an electron-transfer reaction.

In an embodiment, the controller controls the ion optics and detector soas to:

-   -   (d) isolate a range of mass-to-charge ratios of the distribution        of mass-to-charge-manipulated precursor ions, thereby generating        isolated mass-to-charge-manipulated precursor ions; wherein the        range of mass-to-charge ratios of the distribution of        mass-to-charge-manipulated precursor ions corresponding to 100        m/z units or less.

In an embodiment, the controller controls the ion optics and detector soas to:

-   -   (d) isolate a range of mass-to-charge ratios of the distribution        of mass-to-charge-manipulated precursor ions, thereby generating        isolated mass-to-charge-manipulated precursor ions; wherein the        range of mass-to-charge ratios of the distribution of        mass-to-charge-manipulated precursor ions corresponding to 3 m/z        units or less.

In an embodiment, the controller controls the ion optics and detector soas to:

-   -   (d) isolate a range of mass-to-charge ratios of the distribution        of mass-to-charge-manipulated precursor ions, thereby generating        isolated mass-to-charge-manipulated precursor ions; wherein the        range of mass-to-charge ratios of the distribution of        mass-to-charge-manipulated precursor ions corresponding to 1 m/z        units or less.

In an embodiment, the controller controls the ion optics and detector soas to:

-   -   (d) isolate a range of mass-to-charge ratios of the distribution        of mass-to-charge-manipulated precursor ions, thereby generating        isolated mass-to-charge-manipulated precursor ions; wherein the        range of mass-to-charge ratios of the distribution of        mass-to-charge-manipulated precursor ions corresponding to 0.2        m/z units or less.

In an embodiment, the controller controls the ion optics and detector soas to:

-   -   (d) isolate a range of mass-to-charge ratios of the distribution        of mass-to-charge-manipulated precursor ions, thereby generating        isolated mass-to-charge-manipulated precursor ions; wherein the        range of mass-to-charge ratios of the distribution of        mass-to-charge-manipulated precursor ions corresponding to 0.1        m/z units or less.

In an embodiment, the system further comprises a second ion detector incommunication with the first ion separation optics for detecting ionsseparated according to their mass-to-charge ratios and generating firstprecursor ion mass spectrometry data corresponding to the firstdistribution of precursor ions.

In an embodiment, the system further comprises a fourth ion detector incommunication with the ion reaction optics for detecting themass-to-charge ratios of at least a portion of themass-to-charge-manipulated precursor ions, thereby generatingmass-to-charge-manipulated precursor ion mass spectrometry datacorresponding to the distribution of mass-to-charge-manipulatedprecursor ions.

In an embodiment, the system further comprises a fifth ion detector incommunication with the second ion separation optics for detecting ionsseparated according to their mass-to-charge ratios and generating secondprecursor ion mass spectrometry data corresponding to the seconddistribution of precursor ions.

In an embodiment, the controller controls the ion optics and detector soas to:

-   -   (e) fragment ions corresponding to the isolated        mass-to-charge-manipulated precursor ions, thereby generating        first product ions; wherein the isolated        mass-to-charge-manipulated precursor ions are fragmented by        beam-type collisionally activated dissociation, ultraviolet        photo-dissociation, infrared photodissociation, electron        transfer dissociation, electron capture dissociation, surface        induced dissociation, or resonant excitation collisionally        activated dissociation.

In an embodiment, the controller controls the ion optics and detector soas to:

-   -   (j) fragment ions corresponding to the second distribution of        precursor ions, thereby generating second product ions; wherein        the isolated mass-to-charge-manipulated precursor ions are        fragmented by beam-type collisionally activated dissociation,        ultraviolet photo-dissociation, infrared photodissociation,        electron transfer dissociation, electron capture dissociation,        surface induced dissociation, or resonant excitation        collisionally activated dissociation.

In an embodiment, the controller controls the reaction conditions andion optics to maximize the amount of first product ions generated. In anembodiment, the controller controls the reaction conditions and ionoptics to maximize the amount of second product ions generated. In anembodiment, at least a portion of the first product ion massspectrometry data corresponds to a reporter tag of the isobaricallylabeled analyte. In an embodiment, at least a portion of the secondproduct ion mass spectrometry data corresponds to a sequence tag of theisobarically labeled analyte.

In an embodiment, the system further comprises an ion storage device incommunication with the ion fragmentation optics for storing fragmentedions. In an embodiment, the controller further controls the ion opticsand detectors so as to:

-   -   (l) store the first product ions and the second product ions in        the ion storage device coincidentally before measuring the        mass-to-charge ratios of the first product ions and the second        product ions.

In an embodiment, at least a portion of the first product ion massspectrometry data corresponds to an amount of a component of theisobarically labeled analyte. In an embodiment, at least a portion ofthe second product ion mass spectrometry data corresponds to an aminoacid sequence of a component of the isobarically labeled analyte.

In an embodiment, the ion source is an electrospray ionization source ora MALDI source.

In an embodiment, the isobarically labeled analyte comprises proteins orpeptides. In an embodiment, the isobarically labeled analyte comprisesphosphorylated proteins or peptides. In an embodiment, the isobaricallylabeled analyte comprises co-translationally modified proteins orpeptides. In an embodiment, the isobarically labeled analyte comprisespost-translationally modified proteins or peptides. In an embodiment,the isobarically labeled analyte comprises small molecules,pharmaceutical compounds, oligonucleotides, or sugars.

In an embodiment, the system further comprises a fractionation stageoperably connected to the ion source for fractionating the isobaricallylabeled analyte prior to generating the distribution of precursor ionsfrom the isobarically labeled analyte.

In an embodiment, the system comprises a tandem mass spectrometerinstrument or a multistage mass spectrometer instrument.

In an embodiment, the isobarically labeled analyte comprises proteins orpeptides and the controller controls the ion optics and detectors toanalyze the isobarically labeled analyte to quantify the amount ofproteins or peptides in the analyte. In an embodiment, the isobaricallylabeled analyte comprises one or more proteins. In an embodiment, theone or more proteins are digested. In an embodiment, the controlleridentifies peptides corresponding to the one or more proteins. In anembodiment, the controller determines amounts of the one or moreproteins. In an embodiment, the controller determines a composition ofthe one or more proteins. In an embodiment, the controller determines apost-translational modification of the one or more proteins. In anembodiment, the one or more proteins are indicative of a disease state.

In an embodiment, the controller further controls the ion optics anddetector so as to identify the range of mass-to-charge ratios of thesecond distribution of precursor ions to not include mass-to-chargeratios corresponding to reporter ions of the isobarically labeledanalyte. In an embodiment, the identified range of mass-to-charge ratiosof the second distribution of precursor ions does not include ionshaving a mass-to-charge ratio less than 400 m/z units. In an embodiment,the identified range of mass-to-charge ratios of the second distributionof precursor ions does not include ions having a mass-to-charge ratioless than 200 m/z units. In an embodiment, the identified range ofmass-to-charge ratios of the second distribution of precursor ions doesnot include ions having a mass-to-charge ratio less than 175 m/z units.In an embodiment, the identified range of mass-to-charge ratios of thesecond distribution of precursor ions does not include ions having amass-to-charge ratio less than 150 m/z units.

Without wishing to be bound by any particular theory, there can bediscussion herein of beliefs or understandings of underlying principlesor mechanisms relating to the invention. It is recognized thatregardless of the ultimate correctness of any explanation or hypothesis,an embodiment of the invention can nonetheless be operative and useful.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a flowchart illustrating an overview of steps used in anembodiment of the present invention.

FIG. 2 illustrates different types of ion-ion reactions that can alterthe charge of a precursor ion.

FIG. 3 provides a precursor purity model workflow and example massspectra for a mixed yeast and human sample.

FIG. 4 provides a quantitative accuracy model workflow and example massspectra for a mixed yeast and human sample containing interference.

FIG. 5 provides comparative analysis of precursor purity model andquantitative accuracy model samples using either higher-energy collisiondissociation mass spectrometry analysis (HCD MS²), or mass spectrometryanalysis utilizing gas-phase purification as described in an embodimentof the present invention (referred to in the Figure as “QuantMode”).Panel A provides a distribution of precursor purity as measured usingeither HCD MS² or QuantMode. Panel B provides analysis of quantitativeaccuracy via HCD MS² (left), HCD MS² with filtering (middle), andQuantMode (right).

FIG. 6 provides plots showing analysis of the quantitative accuracymodel sample for true ratios of 5:1 (panel A), 3:1 (panel B), 2:1 (panelC) and 1.5:1 (panel D).

FIG. 7 provides a plot showing quantitative accuracy as a function ofprecursor isolation width.

FIG. 8 provides an overview of steps in the QuantMode scan function.

FIG. 9 provides an overview of QuantMode as applied to the quantitativeaccuracy model.

FIG. 10 provides a plot showing differences in optimal collisionenergies for identification and quantification.

FIG. 11 provides a plot illustrating that a number of replicate analysisare often required to detect statistically significant proteindifferences (panel A). Panel B shows multiple measurements that revealsubtle but significant (P<0.05) differential regulation of twophosphorylation sites on NSUN2 that was only detectable with multipleindependent comparisons.

FIG. 12 provides a comparison of precursor purity observed in anisobaric tag experiment without proton transfer ion-ion reactions (PTR)vs. purity distribution following PTR (panel A). Panel B showsquantitative accuracy for an isobaric tagging experiment of yeastpeptides mixed in a 1:10 ratio and contaminated with human interferenceat 1:1.

FIG. 13 provides a plot of the measured isobaric tag ratio as a functionof detectable precursor interference in the MS isolation window (panelA). Even precursors having highly pure (>25%) target precursors havemeasured ratios much lower than expected (dotted line). Panel B showsisobaric tag ratios as a function of target precursor intensity. Targetsof high intensity provide ratios closest to the true value (dottedline).

FIG. 14 provides isobaric tag ratios as a function of reporter tagintensity (panel A). Tags of high intensity provide ratios closest tothe true value (dotted line). Panel B shows isobaric tag ratios as afunction of precursor charge state. Tags of high charge states provideratios furthest from the true value (dotted line).

FIG. 15 provides a spectrum of an isolation window showing an isotopiccluster of a peptide precursor cation of a peptide having the sequenceRINELTLLVQK. Interfering species are present.

FIG. 16 provides an MS/MS spectrum of the precursor region shown in FIG.14. Reporter tags at m/z 126 and 127 should have ratio of 10:1. They aremeasured at 2.5:1.

FIG. 17 provides a spectrum of the reaction of the isolation windowshown in FIG. 14. Multiple products are detected from both the targetpeptide and contaminating species.

FIG. 18 provides a spectrum of an isolation window showing a purifiedprecursor peptide cation population following a proton transfer reactionof the isolation window shown in FIG. 14.

FIG. 19 provides a product ion spectrum following dissociation of theisolated charge-reduced target precursor population. The reporter signalis now measured at 9.9:1 and is almost identical to the true value of10:1.

FIG. 20 provides a product ion spectrum following isolation anddissociation of the charge-reduced precursor of the peptide having thesequence TASGNIIPSSTAGAAK.

FIG. 21 provides a product ion spectrum following (a) isolation anddissociation of the charge-reduced precursor of the peptide having thesequence TASGNIIPSSTAGAAK, and (b) fragmentation of the isolated noncharge-reduced precursor and combination with the products generated instep 1. Significantly more sequence informative ions are present in thisspectrum.

FIG. 22 provides a schematic diagram of an algorithm illustrating thatan embodiment of the present invention can be selectively applied to asample depending on whether highly intense or highly purified precursorsare present.

FIG. 23 provides a flowchart illustrating the sequence of scan events inone embodiment of the present invention (low-res QuantMode) including acharge-state determination scan, one or more quantitation scans, and anidentification scan.

FIG. 24 provides data evaluating charge determination scans performed inan embodiment of the present invention. This data confirms that atargeted charge state scan prediction of a +2 or +3 charged precursorwas correct at least 88% of the time (FIG. 23 a). However, the targetedscan was only able to predict charge state information for 42% of allprecursors sampled, and only 60% of these predicted charge-states wereassociated with peptides amenable to PTR (≧+2 charge) (FIG. 23 b). Thetargeted scan also generated significantly less predictions for the +3charged precursors (FIG. 23 c). FIG. 23 d shows that integration of thetargeted charge determination scan into the low-res QuantMode scanenabled a shorter duty cycle and a greater amount of peptideidentifications than quantitatively evaluating each peptide as a +2, +3,and +4 charged species.

FIG. 25 shows a charge-state distribution of isobarically labeledpeptides where a significant amount of peptides have a charge state of+3, +4 and greater +4.

FIG. 26 shows optimal activation conditions and optimal collision energyfor peptide identification and reporter tag intensity generation.

FIG. 27 shows quantitative results for yeast peptides identified inexperiments utilizing a low-res QuantMode embodiment of the presentinvention compared to mass spectrometry analysis using trapHCD.

FIG. 28A-C show relative protein levels present in the myogenic cells atday 0 and day 6 of the differentiation process in experiments utilizinga low-res QuantMode embodiment of the present invention compared to massspectrometry analysis using trapHCD. FIGS. 27A-C show that fHCD-onlyanalyses were able to identify a greater overall number of proteins thanlow-res QuantMode analyses but that low-res QuantMode actuallyidentified more 1.5-fold changes than fHCD.

FIG. 29 provides spectra for two proteins discovered during a six daytime course. The results from the low-res QuantMode experiment indicatedan 8.0 fold difference in amount of the protein from day zero to day 6,while the trapHCD experiment only indicated a 3.1 fold increase in theprotein.

DETAILED DESCRIPTION

Referring to the drawings, like numerals indicate like elements and thesame number appearing in more than one drawing refers to the sameelement. In general the terms and phrases used herein have theirart-recognized meaning, which can be found by reference to standardtexts, journal references and contexts known to those skilled in theart. The following definitions are provided to clarify their specificuse in the context of the invention.

DEFINITIONS

As used herein, the term “precursor ion” is used herein to refer to anion which is produced during ionization stage of mass spectrometryanalysis, including the MS¹ ionization stage of MS/MS analysis.

As used herein, the terms “product ion” and “secondary ion” are usedinterchangeably in the present description and refer to an ion which isproduced during a fragmentation process of a precursor ion. The term“secondary product ion” as used herein refers to an ion which is theproduct of successive fragmentations.

As used herein, the term “analyzing” refers to a process for determininga property of an analyte. Analyzing can determine, for example, physicalproperties of analytes, such as mass or atomic or substituentcomposition.

As used herein, the term “analyte” refers to a compound, mixture orcomposition which is the subject of an analysis. Analytes include, butare not limited to, proteins, peptides, small molecules, pharmaceuticalcompounds, oligonucleotides, sugars and mixtures thereof. An“isobarically labeled analyte” refers to an analyte that has beenlabeled with one or more isobaric tagging reagents. For example, an“isobarically labeled analyte” can be a mixture containing proteins orpeptides labeled with multiple isobaric tagging reagents where theisobaric tagging reagents generate different reporter ions duringfragmentation.

As used herein, the term “ion source” refers to a device component whichproduces ions from a sample. Examples of ion sources include, but arenot limited to, electrospray ionization sources and matrix assistedlaser desorption/ionization (MALDI) sources.

As used herein, the term “mass spectrometry” (MS) refers to ananalytical technique for the determination of the elemental compositionof an analyte. Mass spectrometric techniques are useful for elucidatingthe chemical structures of analytes, such as peptides and other chemicalcompounds. The mass spectrometry principle consists of ionizing analytesto generate charged species or species fragments and measurement oftheir mass-to-charge ratios. Conducting a mass spectrometric analysis ofan analyte results in the generation of mass spectrometry data relatingto the mass-to-charge ratios of the analyte and analyte fragments. Massspectrometry data corresponding to analyte ion and analyte ion fragmentsis presented in mass-to-charge (m/z) units representing themass-to-charge ratios of the analyte ions and/or analyte ion fragments.In tandem mass spectrometry (MS/MS or MS²), multiple rounds of massspectrometry analysis are performed. For example, samples containing amixture of proteins and peptides can be ionized and the resultingprecursor ions separated according to their mass-to-charge ratio.Selected precursor ions can then be fragmented and further analyzedaccording to the mass-to-charge ratio of the fragments.

As used herein, the term “interference” refers to a species detected inan analysis which interferes with the detection of a species or analyteof interest. Interference can refer to detection of a protein, orprotein fragment, which is not a protein or protein fragment of interestand which interferes with the accurate detection or quantitation of theprotein or peptide fragment of interest. Interference can be quantifiedas an interference ratio, such as a ratio of an amount of interferencesignal to an amount of analyte signal. In a mass spectral analysis,interference can be manifested as an interference peak which correspondsto detection of a species which is not an analyte of interest.

As described herein, an “isolation window” refers to a range ofprecursor ions or a range of mass-to-charge-manipulated precursor ionsthat is selectively separated and fragmented, manipulated or isolated.For example, the range of precursor ions which undergo manipulation oftheir mass-to-charge ratios can be referred to as a first isolationwindow; and the range of mass-to-charge-manipulated precursor ions thatis isolated prior to fragmentation can be referred to as a secondisolation window.

As used herein, the term “species” refers to a particular molecule,compound, ion, anion, atom, electron or proton. For example, asdescribed in certain embodiments herein, precursor ions within aselected mass-to-charge ratio range are reacted with a species to changethe mass-to-charge ratio of the precursor ions. This means the selectedprecursor ions are reacted with a specific molecule, compound, ion,atom, electron or proton to change either the mass of charge of theprecursor ions. In one embodiment, if it is desired to alter the mass ofthe precursor ions, the species can be a molecule or compound whichbinds to the precursor ions adding to the molecular weight of eachprecursor ion. In another embodiment, if it is desirable to alter thecharge of the precursor ions, the species can be a charge manipulationreactant which adds an electron or proton to a precursor ion therebychanging the charge of the precursor ion.

As used herein, the term “signal-to-noise ratio” refers to a measurewhich quantifies how much a signal has been corrupted by noise, orunwanted signal. It can also refer to the ratio of signal power to thenoise power corrupting the signal. A ratio higher than 1:1 indicatesmore signal than noise and is desirable for some applications.

As used herein, the term “mass-to-charge ratio” refers to the ratio ofthe mass of a species to the charge state of a species. The term “m/zunit” refers to a measure of the mass to charge ratio. The Thomson unit(abbreviated as Th) is an example of an m/z unit and is defined as theabsolute value of the ratio of the mass of an ion (in Daltons) to thecharge of the ion (with respect to the elemental charge).

As used herein, the term “ion optic” refers to a device component whichassists in the transport and manipulation of charged particles, forexample ions, by the application of electric and/or magnetic fields. Theelectric or magnetic field can be static, alternating, or can containboth static and alternating components. Ion optical device componentsinclude, but are not limited to, ion deflectors which deflect ions, ionlenses which focus ions, and multipoles (such as quadruples) whichconfine ions to a specific space or trajectory. Ion optics includemultipole RF device components which comprise multiple rods having bothstatic and alternating electric and/or magnetic fields.

As used herein, the term “mass spectrometer” refers to a device whichcreates ions from a sample, separates the ions according to mass, anddetects the mass and abundance of the ions. Mass spectrometers includemultistage mass spectrometers which fragment the mass-separated ions andseparate the product ions by mass one or more times. Multistage massspectrometers include tandem mass spectrometers which fragment themass-separated ions and separate the product ions by mass once.

As used herein, the term “disease state” refers to condition that cancause pain, dysfunction, distress, social problems, and/or death to apatient. Methods and systems described herein can be useful fordiagnosis of a disease state.

The terms “peptide” and “polypeptide” are used synonymously in thepresent description, and refer to a class of compounds composed of aminoacid residues chemically bonded together by amide bonds (or peptidebonds). Peptides and polypeptides are polymeric compounds comprising atleast two amino acid residues or modified amino acid residues.Modifications can be naturally occurring or non-naturally occurring,such as modifications generated by chemical synthesis. Modifications toamino acids in peptides include, but are not limited to,phosphorylation, glycosylation, lipidation, prenylation, sulfonation,hydroxylation, acetylation, methylation, methionine oxidation,alkylation, acylation, carbamylation, iodination and the addition ofcofactors. Peptides include proteins and further include compositionsgenerated by degradation of proteins, for example by proteolyicdigestion. Peptides and polypeptides can be generated by substantiallycomplete digestion or by partial digestion of proteins. Polypeptidesinclude, for example, polypeptides comprising 1 to 100 amino acid units,optionally for some embodiments 1 to 50 amino acid units and, optionallyfor some embodiments 1 to 20 amino acid units.

“Protein” refers to a class of compounds comprising one or morepolypeptide chains and/or modified polypeptide chains. Proteins can bemodified by naturally occurring processes such as post-translationalmodifications or co-translational modifications. Exemplarypost-translational modifications or co-translational modificationsinclude, but are not limited to, phosphorylation, glycosylation,lipidation, prenylation, sulfonation, hydroxylation, acetylation,methylation, methionine oxidation, the addition of cofactors,proteolysis, and assembly of proteins into macromolecular complexes.Modification of proteins can also include non-naturally occurringderivatives, analogues and functional mimetics generated by chemicalsynthesis. Exemplary derivatives include chemical modifications such asalkylation, acylation, carbamylation, iodination or any modificationthat derivatizes the protein.

As used herein, the term “controller” refers to a device component whichcan be programmed to control a device or system, as is well known in theart. Controllers can, for example, be programmed to control massspectrometer systems as described herein. Controllers can be programmed,for example, to carry out ion manipulation and sample analysis methodsas described herein on systems and devices as described herein.

As used herein, the term “fractionated” or “fractionate” refers to thephysical separation of a sample, as is well known in the art. A samplecan be fractionated according to physical properties such as mass,length, or affinity for another compound, among others usingchromatographic techniques as are well known in the art. Fractionationcan occur in a separation stage which acts to fractionate a sample ofinterest by one or more physical properties, as are well known in theart. Separation stages can employ, among other techniques, liquid andgas chromatographic techniques. Separation stages include, but are notlimited to, liquid chromatography separation systems, gas chromatographyseparation systems, affinity chromatography separation systems, andcapillary electrophoresis separation systems.

Quantitative analysis in chemistry is the determination of the absoluteor relative abundance of one, several, or all particular substance(s)present in a sample. For biological samples, quantitative analysisperformed via mass spectrometry can determine the relative abundance ofpeptides and proteins. The accepted methodology for performing mass specquantitation is accomplished using a mass spectrometer capable of MS/MSfragmentation (i.e. triple quadropole or ion trap). The quantitationprocess typically involves isobaric tagging of peptide precursors, whichwhen combined with post-acquisition software, provides the relativeabundance of peptides.

Gas-Phase Purification

Described herein are methods to eliminate interference and to increasepurification during mass spectrometry analysis of isobaric taggedanalytes. Particularly with lower resolution instruments, interferenceof ions having similar mass-to-charge ratios erodes dynamic rangemeasurements and decreases accuracy of quantitative analysis, asmeasured ratios tend to be compressed toward the median (1:1). Asdescribed herein, the mass-to-charge ratios of precursor ions within afirst isolation window are selectively modified and the modifiedprecursor ions within a second isolation window are then selected forfragmentation and further MS analysis. The use of gas-phase purificationcan therefore exclude undesired ions from subsequent MS measurements.

One embodiment of the present invention is illustrated in FIG. 1 whereionization during the MS¹ stage generates a plurality of precursor ions.In a first selection window, precursor ions having m/z values fallingwithin a desired range are selected and their mass-to-charge ratiossubsequently modified to generate modified precursor ions. In a secondselection window, modified precursor ions having modified m/z valuesfalling within a second desired range are selected and undergo MS²fragmentation and analysis. Optionally, a second MS¹ ionization step isperformed to generate a second set of precursor ions. In a thirdisolation window, precursor ions having m/z values falling within athird desired range are selected and undergo subsequent MS²fragmentation and analysis without modification of their mass-to-chargeratios. Optionally, the fragment ions from the modified precursor ionsselected from the second isolation window and the fragment ions from theunmodified precursor ions selected from the third isolation window arecombined for MS² analysis.

In one embodiment, the present invention allows purification andaccurate mass spectrometry analysis of analytes which produce multipleprecursor ions in the MS¹ stage having similar mass-to-charge ratios.These multiple precursor ions having similar mass-to-charge ratios canbe reporter ions from isobarically tagged molecules, such asisobarically tagged peptides. The similarity of the mass-to-chargeratios between multiple precursor ions may also arise because theprecursor ions are generated from closely related peptides. For example,proteins and peptides that undergo various degrees of phosphorylationmay result in precursor ions having very similar mass-to-charge ratios.FIG. 11 displays data detailing the number of biological replicateanalyses required to determine statistically significant differencesbetween human ES and induced pluripotent cell lines (iPS). FIG. 11 showssubtle but significant (P<0.05) differential regulation of aphosphorylation site on NSUN2 that is only detectable with 12independent comparisons. The number of replicate analysis required todetect statistically significant protein differences is shown in FIG. 11(panel A). Panel B of FIG. 11 shows multiple measurements that revealsignificant (P<0.05) differential regulation of two phosphorylationsites on NSUN2. The present invention increases the accuracy of the massspectrometry analysis of such proteins and allows for the determinationof the amount of such proteins.

The mass-to-charge ratios of the precursor ions can be selectivelychanged by reacting the precursor ions, which are typically positivelycharged, with a species (such as a charge manipulation reactant) so asto change the charge, the mass, or both, of the precursor ion. In oneembodiment, the reaction with a species to selectively change themass-to-charge ratios is an ion-ion reaction that at least changes thecharge of the precursor ion. In a further embodiment, the ion-ionreaction is selected from the group consisting of a proton transferreaction (PTR), an electron transfer reaction, and an anion attachmentreaction. For example, these reactions, as illustrated in FIG. 2, reacta positively charged ion (cation) with a negatively charged speciesresulting in a net loss of a charge of the cation. With an electrontransfer reaction, the cation gains an electron from the negativelycharged species. A proton transfer reaction can be described as thecation losing a proton to the negatively charged species. The resultingmass change in an electron transfer and proton transfer reaction isminimal. With an anion attachment reaction, the negatively chargedspecies binds to the cation which noticeably alters the charge and themass of the cation.

The mass-to-charge ratio of the precursor ions can be changed bymanipulating the mass or charge of the precursor ions. For example, evenbefore electron-transfer dissociation (ETD), proton transfer ion-ionreactions (PTR) were developed to manipulate precursor charge states,purify precursors, and simplify spectra. PTR is typically accomplishedby reaction of multiply protonated cations with even-electron anions,resulting in precursor ion de-protonation and charge state reduction. AnETD-enabled orbitrap can permit both ETD and PTR chemistry. Accordingly,one embodiment of the present invention provides a novel approach ofusing PTR to enable large-scale, multiplexed protein quantification.Stable isotope labeling by amino acids in cell culture (SILAC), theprotein quantification gold standard, provides a means to make binary orternary comparisons [See, e.g., Ong, S. E., B. Blagoev, I. Kratchmarova,D. B. Kristensen, H. Steen, A. Pandey, and M. Mann, Stable isotopelabeling by amino acids in cell culture, silac, as a simple and accurateapproach to expression proteomics. Molecular & Cellular Proteomics,2002. 1(5): p. 376-386]. SILAC, however, has two major drawbacks:incompatibility with human tissues or biofluids, clearly the majorportion of biomedical research, and high labor requirements, which makebiological replicate analysis difficult to achieve. The problem is thateach binary or ternary set must be analyzed separately. This issue,combined with extensive fractionation, and the need for technicalreplicates, means that a large-scale experiment via SILAC demands threeto five months of constant instrument usage for a single biologicalreplicate. And though this approach can detect extreme differences, itdoes not afford the statistical power sufficient to reveal subtle oreven moderate perturbations and does not offer control or estimation offalse positive rates.

Isobaric labeling provides a solution to this problem, allowing relativequantification of up to 8 proteomes simultaneously [See: Thompson, A.,J. Schafer, K. Kuhn, S. Kienle, J. Schwarz, G. Schmidt, T. Neumann, andC. Hamon, Tandem mass tags: A novel quantification strategy forcomparative analysis of complex protein mixtures by ms/ms. AnalyticalChemistry, 2003. 75(8): p. 1895-1904; Ross, P. L., Y. L. N. Huang, J. N.Marchese, B. Williamson, K. Parker, S. Hattan, N. Khainovski, S. Pillai,S. Dey, S. Daniels, S. Purkayastha, P. Juhasz, S. Martin, M.Bartlet-Jones, F. He, A. Jacobson, and D. J. Pappin, Multiplexed proteinquantitation in saccharomyces cerevisiae using amine-reactive isobarictagging reagents. Molecular & Cellular Proteomics, 2004. 3(12): p.1154-1169; Phanstiel, D., R. Unwin, G. C. McAlister, and J. J. Coon,Peptide quantification using 8-plex isobaric tags and electron transferdissociation tandem mass spectrometry. Anal Chem, 2009. 81(4): p.1693-8; Choe, L., M. D'Ascenzo, N. R. Relkin, D. Pappin, P. Ross, B.Williamson, S. Guertin, P. Pribil, and K. H. Lee, 8-plex quantitation ofchanges in cerebrospinal fluid protein expression in subjects undergoingintravenous immunoglobulin treatment for alzheimer's disease.Proteomics, 2007. 7(20): p. 3651-3660].

Here, differentially isotopically labeled, but isobaric amine-reactivetags are embedded into peptides. Once labeled, the 8 samples arecombined and peptides are sequenced individually by MS/MS using CAD.Peptides having the same sequence from each of the 8 samples co-eluteand have equivalent m/z values. During MS/MS, however, vibrationalexcitation induces cleavage of both the peptide backbone and theisobaric tag. Dissociation of the backbone gives rise to fragment ionscharacteristic of the peptide sequence; dissociation of the taggenerates low mass product ions where each label creates a unique m/zreporter peak. Yet despite the potential to enable expedient,multiplexed quantitative proteomics, precursor interference hasprevented isobaric labeling from being widely embraced. [See, Lu, R., F.Markowetz, R. D. Unwin, J. T. Leek, E. M. Airoldi, B. D. MacArthur, A.Lachmann, R. Rozov, A. Ma'ayan, L. A. Boyer, O. G. Troyanskaya, A. D.Whetton, and I. R. Lemischka, Systems-level dynamic analyses of fatechange in murine embryonic stem cells. Nature, 2009. 462(7271): p.358-U126]. This problem does not exist for SILAC because abundancemeasurements are performed with high-resolution MS¹. Even for verycomplex samples having tens or hundreds of co-eluting peptides,high-resolving power mass analyzers can easily distinguish the targetfrom neighboring peaks less than 0.01 Th away. In the isobaric approach,however, the target peptide is isolated at much lower resolution,typically 1-3 Th, and dissociated to produce reporter tags. Therefore,the quantitative signal in the reporter region is compiled from everyspecies in the isolation window. [See, Ow, S. Y., M. Salim, J. Noirel,C. Evans, I. Rehman, and P. C. Wright, Itraq underestimation in simpleand complex mixtures: “The good, the bad and the ugly”. Journal ofProteome Research, 2009. 8(11): p. 5347-5355]. For highly complexmixtures, like those analyzed in large-scale experiments, co-isolationof multiple species is the rule, not the exception. This problem mayerode dynamic range in some situations, as measured ratios tend to becompressed toward the median ratio of 1:1, and thus has restricted thetechnique to applications with low sample complexity. [See Karp, N. A.,W. Huber, P. G. Sadowski, P. D. Charles, S. V. Hester, and K. S. Lilley,Addressing accuracy and precision issues in itraq quantitation. Mol CellProteomics, 2010]. Described below are methods utilizing gas-phasepurification, such as through changing the mass-to-charge ratio via PTR,to systematically eliminate the pervasive interference problem inisobaric tag-based quantification. Allowing large-scale comparison of 8or more proteomes simultaneously, this straightforward approach haspotential to transform the way quantitative proteomics is conducted.

Example 1 Gas-Phase Purification Enables Accurate, Large-Scale,Multiplexed Proteomic Quantification

Protein identification technologies have rapidly matured such thatconstructing catalogs of the thousands of proteins comprised by a cellusing mass spectrometry (MS) is now relatively straightforward [deGodoy, L. M. F. et al. Nature 455, 1251-1255 (2008); Swaney, D. L.,Wenger, C. D. & Coon, J. J. J. Proteome Res. 9, 1323-1329 (2010)].Knowing how the abundance of these molecules change under variouscircumstances is not [Ong, S. E. & Mann, M. Nat. Chem. Biol. 1, 252-262(2005)]. Stable isotope labeling by amino acids in cell culture (SILAC)provides a means to make binary or ternary comparisons [Jiang, H. &English, A. M. J. Proteome Res. 1, 345-350 (2002); Ong, S. E. et al.Mol. Cell. Proteomics 1, 376-386 (2002)]. By interlacing these two- orthree-way experiments, higher-order comparisons can be obtained [Olsen,J. V. et al. Sci. Signal. 3, ra3 (2010)]. Such large-scale multiplexedexperiments are invaluable, as they (1) allow measurement of time-courseexperiments, (2) permit collection of biological replicates, and (3)enable direct comparison of transcriptomic and proteomic data.

Constructing this type of multi-faceted proteomics study, however, is anarduous undertaking and has only been accomplished in a handful ofexperiments by an even smaller group of researchers. The firstimpediment is the requirement to grow multiple groups of cells withvarious labels. And this step is actually less limiting than the secondmajor obstacle: each binary or ternary set must be analyzed separately.When combined with the need for extensive pre-MS fractionation andtechnical replicates, a large-scale experiment via SILAC demands threeto six months of constant instrument usage.

Isobaric tagging [Thompson, A. et al. Anal. Chem. 75, 1895-1904 (2003);Ross, P. L. et al. Mol. Cell. Proteomics 3, 1154-1169 (2004)], allowsrelative quantification of up to eight proteomes simultaneously [Choe,L. et al. Proteomics 7, 3651-3660 (2007); Dayon, L. et al. Anal. Chem.80, 2921-2931 (2008)]. Further, it is compatible with mammalian tissuesand biofluids, unlike metabolic approaches. Despite its potential toenable fast, multiplexed quantitative proteomics, isobaric tagging hasnot been widely embraced for large-scale studies [Lu, R. et al. Nature462, 358-U126 (2009)]—chiefly because of precursor interference. Thisproblem does not exist for SILAC because abundance measurements areperformed with high-resolution MS¹. Even for very complex samples havingtens or hundreds of co-eluting peptides, high-resolving power massanalyzers can easily distinguish the target from neighboring peaks lessthan 0.01 Th away.

In the isobaric approach, however, the target peptide is isolated atmuch lower resolution, typically 1-3 Th, and dissociated to producereporter tags. Therefore, the quantitative signal in the reporter regionis compiled from every species in the isolation window [Ow, S. Y. et al.J. Proteome Res. 8, 5347-5355 (2009)]. For highly complex mixtures, likethose analyzed in large scale experiments, co-isolation of multiplespecies is the rule, not the exception (vide infra). This problem erodesquantitative accuracy, as measured ratios tend to be compressed towardthe median ratio of 1:1, and thus has restricted isobaric tagging toapplications with lower sample complexity.

Extent of Interference on Quantitative MS² Measurements

To document the extent of interference, a precursor purity model wasconstructed (shown in FIG. 3) by labeling peptides from a whole cellyeast lysate with the tandem mass tag (TMT) 6-plex m/z 126 tag. Thesepeptides were then spiked with peptides from a tryptic digest of humanproteins, which had been labeled with the TMT 6-plex m/z 131 tag. Byincorporating human peptides as the interference, the precursorcontamination typical of a human proteomic analysis can be effectivelymodeled. Following nHPLC-MS/MS, the reporter m/z regions of MS/MSspectra that were uniquely mapped to yeast were examined. The relativesignal abundance of these m/z peaks thus provides a quantitativeempirical measurement of interference. On average, only 68% of reporterion signal originated from the target peptide, as shown in panel A ofFIG. 5. Only 3% of MS/MS spectra were of ultrapure (≧99%) precursors.

FIG. 4 provides analysis of the precursor purity model and quantitativeaccuracy model samples with either HCD MS² or mass spectrometry analysisutilizing gas-phase purification via PRT (referred to in the Figure as“QuantMode”). Panel A of FIG. 4 shows distribution of precursor purityas measured by examining reporter tag 126 (yeast) and 131 (human) foryeast-identified sequences using either HCD MS2 or QuantMode. Panel B ofFIG. 4 shows analysis of quantitative accuracy via HCD MS² (left), HCDMS² with filtering (middle), and QuantMode (right). The dashedhorizontal line indicates the true ratio while boxplots indicate themedian (stripe), the 25th to 75th percentile (interquartile range, box),1.5 times the interquartile range (whiskers), and outliers (opencircles). The number of quantified yeast PSMs (N) and median ratio aregiven for each method.

To address what effect this prevalent interference has on isobarictag-based quantification, a quantitative accuracy model, shown in FIG.5, was created by labeling yeast peptides with TMT 6-plex tags (m/z126-131) and combining them in ratios of 10:1:3:2:5:1.5. This sample wascombined with an equal mass of tryptic human peptides also labeled withTMT 6-plex (m/z 126-131) but mixed in ratios of 1:1:1:1:1:1. Byexamining reporter ion ratios in yeast peptide spectra, the degradationof quantitative accuracy was assessed. Because this mixture was analyzeddirectly with nanoflow liquid chromatography-MS/MS (nLC-MS/MS), withoutprior fractionation, the experiment tested the technology in aworst-case interference scenario. Shockingly, the yeast 10:1 mix wasmeasured at 4.4:1-a 2.3-fold underestimation or a 66% relative error, asshown in FIG. 6, furthest left boxplot in panel B; other similar ratiosare shown in FIG. 6, panels A-D. These experiments both illuminate theextent and repercussion of precursor interference and provide anexcellent metric with which to gauge improvement.

As shown in FIG. 6 (analysis of the quantitative accuracy model sample),boxplots display results for HCD MS² (left), HCD MS² with filtering(middle) or QuantMode (right) at (a) 5:1, (b) 3:1, (c) 2:1, and (d)1.5:1 ratios. The dashed horizontal line indicates the true ratio whileboxplots indicate the median (stripe), the 25th to 75th percentile(interquartile range, box), 1.5 times the interquartile range(whiskers), and outliers (open circles). The number of quantified yeastPSMs (N) and median ratio are given for each method. As seen in FIG. 6,QuantMode provided significantly more accurate results than both HCD MS²and HCD MS² with filtering.

Effects of Narrowing the Isolation Window and Interference Filtering inHCD MS²

Several strategies have been proposed for the elimination of precursorinterference in isobaric tag quantitation. These methods include:narrowing the precursor isolation window, rejecting precursor isolationwindows containing impurities above a certain threshold, and performingan extra fragmentation (MS³) event on the most intense MS² fragment ionprior to mass analysis of the reporter region (MS³). One possibleapproach to reduce the impact of interference is to simply narrow thewidth of the MS² isolation window (without modifying any of themass-to-charge ratios) so that fewer contaminant ions are present duringprecursor activation. Application of this concept, however, producedonly minor improvements. It should also be noted that isolationefficiency is reduced as window widths narrow, resulting in feweridentifications (nearly 50% from 3 to 1 Th; 3,348 to 1,723 yeast PSMs).

As shown in FIG. 7 (quantitative accuracy as a function of isolationwidth), the 10:1 ratio of the quantitative accuracy model sample wasanalyzed by HCD MS² with decreasing precursor isolation width. Thedashed horizontal line marks the true ratio of each plot (10:1). Asshown in FIG. 7, the width of the isolation window (which only selectedprecursor ions within 1 Th to 3 Th mass-to-charge ratios of the targetion) was compared at widths of 3 Th, 2.5 Th, 2.0 Th, 1.5 Th and 1.0 Th.However, the 10:1 mix was still only measured at 4.3:1 to 5.4:1 usingHCD MS². The number of yeast PSMs (n) and median ratio are listedbeneath their respective boxplot.

Another strategy is to reject quantitative information from precursorshaving interference above a specified threshold. For the above dataset,for example, a post-acquisition filtering algorithm was used to removeMS/MS spectra if the precursor's purity was below 75% within the 3 ThMS² isolation window of the preceding MS¹ spectrum. As shown in FIG. 4,middle boxplot of panel B, this technique marginally improvesquantification (to 6.2:1), but comes at the expense of 66% of the data:3,098 versus 1,068 quantified PSMs. Though performing this process inreal time boosts quantifiable PSMs, the improvement in quantificationremains subtle. These data evince that background contaminants are notalways detectable in MS¹. It is concluded that none of theseavoidance-based strategies are wholly satisfactory and that interferenceis an omnipresent quantitative limitation.

Gas-Phase Purification Using Proton Transfer Ion-Ion Reactions(PTR)—QuantMode Scan Function

A fresh approach is to combat interference directly via gas-phasepurification: that is, to de-convolve the co-isolated contaminants fromthe precursor in m/z space by manipulating either mass or charge beforeperforming a second isolation. Expedient proton-transfer ion/ionreactions (McLuckey, S. A. & Stephenson, J. L. Mass Spectrom. Rev. 17,369-407 (1998); Reid, G. E., Shang, H., Hogan, J. M., Lee, G. U. &McLuckey, S. A. J. Am. Chem. Soc. 124, 7353-7362 (2002)) efficientlyreduce ion charge state and integrate easily into higher orderinstrument scan methods. Consider a doubly charged precursor at 500 Thco-isolated with a triply charged contaminant also at 500 Th. FollowingPTR, the precursor is now positioned at m/z 999 (+1 charge state), whilethe interfering species is moved to m/z 749.5 (+2 charge state).Subsequent isolation of m/z 999 yields a purified precursor populationfrom which to generate accurate quantification. Contaminants having zidentical to the precursor are still spread in m/z space; for example, a+2 interference at m/z 501 will be transformed to m/z 1001 andeffectively eliminated from the second isolation window (997.5-1000.5).

To evaluate this idea, a scan function was devised comprising thefollowing steps and automated by instrument firmware code modificationas shown in FIG. 8: (1) first cation injection into a quadrupole linearion trap (QLT), (2) first cation precursor isolation (also in the QLT),(3) anion injection (QLT), (4) proton transfer ion-ion reaction (QLT),(5) charge-reduced precursor isolation (QLT), (6) higher-energycollision dissociation (HCD) of the charge-reduced precursor (within theHCD cell), (7) transfer of HCD products (into the c-trap), (8) secondcation injection (into the QLT), (9) second cation precursor isolation(QLT), (10) resonant-excitation collision-activated dissociation (CAD)(QLT), (11) transfer CAD products (c-trap), (12) transfer HCD/CADproducts (orbitrap), and (13) mass analysis of reporter and sequenceions together (orbitrap). This mass spectrometry method using PRT wasnamed QuantMode. The typical QuantMode scan is ˜64% longer than atypical HCD scan, although markedly shorter than a full MS³ experiment(˜115% longer, not including a separate CAD scan for optimalidentification).

FIG. 9 provides an overview of the QuantMode scan function as applied tothe quantitative accuracy model. A triply charged precursor at m/z595.72 was isolated with a 3 Th window (upper left). The precursorisotopic cluster (▾) occupies only 49% of the total ion current in thisregion. QuantMode begins with PTR (step 1); isolation of thecharge-reduced precursor (+2) purifies this target to 85% (step 2); HCDconverts these purified precursors to reporter ions (step 3);resonant-excitation CAD follows re-injection/reisolation of the triplycharged precursor (step 4). The HCD and CAD products are combined in thec-trap for orbitrap mass analysis (lower right panel). Juxtaposedagainst QuantMode is the conventional HCD MS² scan for this impureprecursor (lower left panel). Lower middle insets display the reporterion region (identical intensity scale) and the quantitative accuracyachieved by both approaches for the 10:1 ratio ().

FIG. 9 displays the purifying effects of QuantMode. Examination of the 3Th precursor isolation window (m/z 595.72, +3) from the preceding MS¹scan reveals extensive contamination—merely 49% purity, as shown in FIG.9, upper left panel. This impurity does not hinder sequenceidentification upon HCD, shown in FIG. 9, bottom left panel(RINELTLLVQK, OMSSA expectation value 2×10⁻¹⁰). It does, however,cripple quantitative accuracy: the 10:1 true value is recorded as 2.5:1,see FIG. 9, bottom center left panel. A 30 ms PTR step on this impurepopulation effectively de-convolves the target with high efficiency(˜45% for +3→+2), see FIG. 9, top middle panel. The doubly protonatedprecursor (893.08 Th) is then isolated from the contaminants (85%purity; see FIG. 9, top right panel); note the mass analysis followingPTR, while illustrative, is not necessary, as the precursor'scharge-reduced m/z is easily calculated. This untainted charge-reducedprecursor population is then dissociated under HCD conditions favorableto reporter ion generation, the products of which are stashed in thec-trap. Next, sequence-informative products are produced throughre-injection of the original triply charged precursor (m/z 595.72),isolation, and fragmentation in the QLT. After combination in thec-trap, the mixed ion population is mass analyzed in the orbitrap, seeFIG. 9, bottom right panel. The QuantMode scan, like its HCD-onlycounterpart, yields a high-confidence match to the peptide sequenceRINELTLLVQK (OMSSA expectation value 2×10⁻⁸). Yet in stark contrast tothe truncated 2.5:1 ratio, QuantMode obtains a 9.9:1 ratio virtuallyequivalent the expected 10:1, see FIG. 9, bottom right center panel.

The QuantMode scan affords manifold benefits in presenting theopportunity to conveniently decouple sequence and reporter iongeneration. When dissociation conditions best facilitate reporter iongeneration, spectral IDs suffer from poor sequence ion production, asshown in FIG. 10. First, then, independent control of dissociationparameters can simultaneously improve quantitative accuracy and spectralIDs. Second, decoupling permits the use of isobaric tagging withdissociation methods that are otherwise incompatible (e.g.,resonant-excitation CAD, ECD/ETD, etc.) [Phanstiel, D., Zhang, Y.,Marto, J. A. & Coon, J. J. J. Am. Soc. Mass Spectrom. 19, 1255-1262(2008)]. Third, beyond decoupling benefits, QuantMode combines multipledisparate steps into a single scan to effectively reduce duty cycle.

As shown in FIG. 10 (differences in optimal collision energies foridentification and quantification), a neat sample of trypsin-digestedyeast proteins were analyzed at normalized collision energies (NCE)ranging from 30 to 70. The optimal number of target PSMs at 1% FDR(filled circles) are achieved at an NCE of 40, while the median totalreporter tag intensity (open circles) peaks at 65 NCE.

To validate the efficacy of this method, the mixed organism models werereanalyzed with QuantMode. With the precursor purity model, purityimproved from 68% to 88% among the same 1,297 precursors. Even morestriking is the surge in ultrapure (≧99%) precursors—from 3% to 23%.Furthermore, this enhanced purity translated to quantitative accuracy.Applying QuantMode to analyze the quantitative accuracy model shiftedthe measured ratio from 4.4:1 with HCD to 8.5:1, much nearer to the truevalue of 10:1, as shown in FIG. 4, right boxplot of panel B. Otherratios are similarly present in FIG. 6. Unlike the avoidance-basedstrategies, such as narrowing the isolation window, QuantMode incursonly a minimal loss (21%, 3,098 to 2,459) in identified PSMs—all ofwhich are now suitable for quantification.

In sum, these experiments demonstrate that isobaric tagging suffers fromsystemic loss of quantitative accuracy on account of pervasive andinherent precursor interference. The data acquisition method, QuantMode,described above mitigates this problem through gas-phase purification.QuantMode substantially increases quantitative accuracy without severelypenalizing quantifiable identifications. In a non-laborious fashion,this method facilitates the critically important measurement of proteinand PTM dynamics and performance of biological replicates for properstatistical treatment. Note this initial implementation will doubtlessevolve to include a repertoire of dissociation methods, to improve bothsequence and reporter ion generation, and to lessen duty cycle.Accordingly, QuantMode will render isobaric tagging a viable option foraccurate, large-scale, multiplexed quantification.

Sample Preparation

For the above experiments, the following samples were prepared.Wild-type yeast (Saccharomyces cerevisiae) was grown in rich media to anOD₆₀₀ of 0.6. Cells were harvested and centrifuged for 10 min at 4° C.The resulting cell pellet was washed twice with sterile water andcentrifuged at 1,088×g for 5 min. Lysis buffer of approximately threetimes the cell pellet volume was added. The Lysis buffer contained 8 Murea, 75 mM NaCl, 50 mM tris (pH 8), 10 mM sodium pyrophosphate, 100 mMsodium butyrate, complete mini ETDA-free protease inhibitor (RocheDiagnostics), and phosSTOP phosphatase inhibitor (Roche Diagnostics).The cells were lysed using a French press where the sample was pressed 3times and centrifuged for 15 min at 23,708×g and 4° C.

Human H1 embryonic stem cells were maintained in a feeder-independentsystem, as previously described [Ludwig, T. E. et al. Nat. Methods 3,637-646 (2006)]. Cells were harvested by individualizing for 10 min withan adequate volume of pre-warmed (37° C.) 0.05% Tryp-LE to cover theculture surface. Following cell detachment, an equivalent volume ofeither icecold DPBS (Invitrogen) was added before collecting the cells.Cell pellets were subsequently washed twice in ice-cold PBS and storedat −80° C. Cells were lysed via sonication in lysis buffer containing 30mM NaCl, 50 mM tris (pH 8), 2 mM MgCl₂, 50 mM NaF, 1 mM sodiumorthovanadate, 6 mM sodium pyrophosphate, mini EDTA-free proteaseinhibitor (Roche Diagnostics), and phosSTOP phosphatase inhibitor (RocheDiagnostics).

For both yeast and human proteins, cysteine residues were reduced withDTT, alkylated using iodoacetamide, and digested in a two-step process(separately). Proteinase Lys-C (Wako Chemicals) was added at anenzyme:protein ratio of 1:100 and incubated for approximately 4 hr at37° C. in lysis buffer. Samples were then diluted with 50 mM tris (pH8.0) to a final urea concentration of 1.5 M and digested withsequencing-grade trypsin (Promega) at an enzyme:protein ratio of 1:50 at37° C. overnight. Reactions were quenched using trifluoroacetic acid.Samples were desalted using C18 solidphase extraction columns (SepPak;Waters) and dried to completion. Yeast and human peptides were thensplit into six equal mass aliquots. TMT labeling was then performed witheach of these aliquots, independently, as described previously [Wenger,C. D., Phanstiel, D. H., Lee, M. V., Bailey, D. J. & Coon, J. J.Proteomics (2011)].

As shown in FIG. 3 (precursor purity model workflow), two populations ofcells, yeast and human, were lysed and enzymatically digested withtrypsin. Yeast peptides labeled with TMT 6-plex m/z 126 tag were mixedin equal mass with human peptides labeled with TMT 6-plex m/z 131 tag.Examining the presence of m/z 131 in yeast-exclusive peptidesfacilitates a quantitative calculation of precursor purity. As shown inFIG. 5 (quantitative accuracy model workflow), two populations of cells,yeast and human, were lysed and enzymatically digested with trypsin.Yeast peptides labeled with TMT 6-plex m/z 126-131 tags in ratios of10:1:3:2:5:1.5, respectively, were mixed in equal mass with humanpeptides labeled with TMT 6-plex m/z 126-131 tags in ratios of1:1:1:1:1:1. Including human peptide contamination in a yeast-exclusivePSM erodes quantitative accuracy.

The precursor purity sample shown in FIG. 3 comprised equal masses of ayeast aliquot labeled with the TMT 6-plex m/z 126 tag and a humanaliquot labeled with the TMT 6-plex m/z 131 tag. The quantitativeaccuracy sample, shown in FIG. 5, comprised six yeast and six humanaliquots labeled with TMT 6-plex m/z 126-131 tags. The yeast aliquotswere mixed in the mass ratios 10:1:3:2:5:1.5, respectively; the humanaliquots were mixed in the mass ratios 1:1:1:1:1:1, respectively. Thesesamples were subsequently combined in equal masses.

Liquid Chromatography-Mass Spectrometry.

Online chromatography was performed with a NanoAcquity UPLC system(Waters) coupled to an ETD-enabled LTQ Orbitrap Velos (ThermoScientific). Samples were loaded onto a precolumn (75 μm i.d., packedwith 10 cm 5 μm Magic C18 particles; Michrom Bioresources, Inc.) for 10min at 98:2 0.2% formic acid:acetonitrile with 0.2% formic acid at aflow rate of 1 μL/min. Samples were then eluted over an analyticalcolumn at a flow rate of 250 mL/min (50 μm i.d., packed with 25 cm 5 μmMagic C18 particles; Michrom Bioresources, Inc.) using a gradient withan initial steep rise to 8% B (acetonitrile with 0.2% formic acid)within 5 minutes, followed by a linear gradient to 30% B over 120minutes and a ramped up to 70% B over 10 minutes and held for 5 minutes.The column was equilibrated with 2% B for an additional 25 min. Thecolumn-making procedure was previously described [Martin, S. E.,Shabanowitz, J., Hunt, D. F. & Marto, J. A. Anal. Chem. 72, 4266-4274(2000)].

Unless otherwise noted, the MS instrument method consisted of adata-dependent top-10 experiment with MS¹ resolution of 30,000(orbitrap) followed either HCD MS² or QuantMode scans, mass analyzed inthe orbitrap at 7,500 resolution. All isolation widths were 3 Th. HCDused a NCE setting of 45 for 30 ms. QuantMode employed PTR, the anionsfor which were generated by the commercial ETD source. For best PTRperformance, we employed the nitrogen adduct of fluoranthene at m/z 216.Source conditions were optimized and all associated ion optics, usingthe instrument's automated tuning procedures, for this PTR anion.

Following accumulation of the precursor and reagent ions, the twopopulations were mixed via charge-sign independent trapping, as in anETD scan. Reaction conditions were tailored to produce the maximalamount of the single charge reduction—e.g., the ion/ion reaction timefor a triply charged precursor was set such that the maximum amount ofdoubly charge product ion was produced. For these experiments thefollowing reaction times were employed: doubly charged precursors werereacted for 80 ms, triply charged precursors were reacted for 30 ms,quadruply charged precursors were reacted for 20 ms, and all precursorswith five or more charges were reacted for 10 ms.

Following PTR, the resulting charge-reduced population was isolated andsubjected to HCD (NCE 45, 30 ms). Note the charge-reduced species wasused for determining HCD energetics, not the initial precursor. Theseproducts are then stored in the c-trap. Following HCD, but prior to massanalysis in the orbitrap, a second population of precursor ions (notcharge reduced, i.e., impure) was injected into the high-pressure QLT,isolated, and subjected to resonant-excitation CAD (q-value 0.25, NCE35, 10 ms). These products, which are devoid of the reporter region onaccount of the QLT low mass cutoff, were mixed with the HCD products inthe c-trap and then simultaneously mass analyzed in the orbitrap.

AGC target values were 1,000,000 (MS¹), 50,000 (HCD), 200,000(QuantMode: PTR), and 10,000 (QuantMode: resonant-excitation CAD). Notemore recent data indicates a reduction in QuantMode PTR target to 50,000results in neither significant loss of quantitative accuracy noridentification rate. Precursors of unknown or +1 charge state wererejected. Dynamic exclusion was enabled for 60 s after one precursorselection.

For the QuantMode overview experiment shown in FIG. 9, a data-dependenttop-3 instrument method consisting of a 30,000-resolution MS¹ scan inthe orbitrap was used followed by (1) normal HCD MS², (2) PTR MS², and(3) HCD MS³ of the charge-reduced precursor followed by CAD MS² of theoriginal precursor, in three separate scans, all mass analyzed at 7,500resolution in the orbitrap. For the isolation width experiments shown inFIG. 7, standard HCD was used with MS² isolation widths from 3 Th to 1Th in increments of 0.5 Th. For HCD NCE experiments shown in FIG. 10,standard HCD was used with NCEs of 30 to 70 in increments of 5. In thiscase only the yeast component of the quantitative accuracy sample wasused with no human interference.

Data Analysis

The resulting data was processed with the COMPASS software suite[Wenger, C. D., Phanstiel, D. H., Lee, M. V., Bailey, D. J. & Coon, J.J. Proteomics (2011)]. OMSSA [Geer, L. Y. et al. J. Proteome Res. 3,958-964 (2004)] searches were performed against a target-decoy [Elias,J. E. & Gygi, S. P. Nat. Methods 4, 207-214 (2007)] database containingboth human (International Protein Index, 3.80) and yeast (SaccharomycesGenome Database, www.yeastgenome<dot>org, Feb. 3, 2011, “all” versionincluding all systematically named open reading frames (ORFs), includingverified, uncharacterized, and dubious ORFs and pseudogenes) proteinsusing an average precursor mass tolerance of ±5.0 Da and a monoisotopicproduct mass tolerance of ±0.01 Da. Carbamidomethylation of cysteines(+57 Da), TMT 6-plex on peptide N-termini (+229 Da) and TMT 6-plex onlysines (+229 Da) were specified as fixed modifications. Oxidation ofmethionine (+16 Da) and TMT 6-plex on tyrosines (+229 Da) were specifiedas variable modifications.

All analyses were independently filtered to 1% FDR at the unique peptidelevel. Peptides that could be derived from human proteins, regardless ofenzymatic specificity and treating leucine and isoleucine as equivalent,were removed from consideration so only peptides of yeast origin wereevaluated for quantitative analysis.

Post-acquisition precursor purity assessment was performed in real timewith additional logic to the instrument firmware. The algorithm consistsof iterating through all peaks within a window around the precursor. Thewindow was enlarged by 20% relative to the actual isolation window, from3 Th to 3.6 Th, to account for the empirical observation that speciesoutside the isolation window (particularly on the low m/z side) couldstill be retained at significant levels. The peak m/z was converted tomass and compared to the precursor mass, assuming both species had thesame charge. The nearest multiple of 1.00335 Da (carbon-13 mass minuscarbon-12 mass, the main contributor to peptide isotopic peaks) wassubtracted, and the remaining mass error was converted to parts permillion (ppm). If the mass error was greater than ±25 ppm, the peak wasjudged as a precursor peak and its intensity was added to the precursorintensity sum. Once all peaks were considered, precursor purity wascalculated as the precursor intensity sum divided by the totalintensity.

Example 2 The Role of Gas-Phase Purification in Quantitative Proteomics

The present methods are useful for providing mass spectrometric analysisachieving a number of goals particularly useful for the analysis ofprotein containing samples. First, the pervasive interference thatoccurs during isobaric tagging with proton transfer ion-ion reactions iseliminated. Additionally, the present methods are applicable in thecontext of the yeast environmental stress response (ESR). Yeast'smanageable proteome and available copy per cell data make it anexcellent tech development model, while the conserved stress responsenetwork makes it medically relevant.

To maximize biomedical impact, quantitative protoemics should becompatible with human tissues and biofluids. Multiplexing is likewisebeneficial as it presents an expedient route to significance testing.Isobaric tagging meets these requirements, but is plagued by ubiquitousand debilitating interference. Such Interference, often undetectable inMS¹ scanning, is effectively combated by gas-phase purification withPTR. This innovative and creative use of PTR has broad biologicalsignificance and impact. Selection of the yeast ESR application is afresh approach to technology development, as those experiments willgenerate guiding information for the subsequent biological study, whichhas relevance for research ranging from human disease to evolution.

Isobaric tagging offers a direct means to perform highly-multiplexedproteome quantification on ALL proteomic samples, including tissues andbio-fluids. The method, however, has not been widely adopted because ofthe widespread problem of precursor interference. [See Lu, R., F.Markowetz, R. D. Unwin, J. T. Leek, E. M. Airoldi, B. D. MacArthur, A.Lachmann, R. Rozov, A. Ma'ayan, L. A. Boyer, O. G. Troyanskaya, A. D.Whetton, and I. R. Lemischka, Systems-level dynamic analyses of fatechange in murine embryonic stem cells. Nature, 2009. 462(7271): p.358-U126]. Co-isolated species unintentionally contribute to thetarget's reporter ion signal, causing an overall repression of dynamicrange and loss of quantitative precision.

An accurate, multiplexed method for quantitative analysis of celllysates, tissues, and biofluids will greatly improve the biomedicalimpact of proteomics. The present approach seamlessly integrates PTRwith ETD/CAD for accurate, high dynamic range, multiplexedquantification.

Experimental Design

To document the extent of interference, peptides from a whole cell yeastlysate were labeled with a tandem mass tag (TMT) 126. These peptideswere spiked with peptides from a digest of human proteins, which hadbeen labeled with a TMT 131 tag. Following nHPLC-MS/MS, the MS/MSreporter regions of peptide precursors that were uniquely mapped toyeast were extracted. The 126:131 reporter signal ratio, therefore,provides a direct measure of contamination, as shown in FIG. 12, panelA. From these data, on average, only 55-60% of reporter region ionsignal originates from the peptide being quantified. The impact ofcontamination on dynamic range is nothing short of astonishing, as shownin FIG. 12, panel B. Yeast peptides labeled either with TMT 126 or 127are mixed in a 1:10 ratio. That sample was contaminated with humanpeptides carrying the same tags, but mixed in a 1:1 ratio. Now anyyeast-identified peptide having human contamination will display aneroded 1:10 ratio. From FIG. 12, panel B (unfiltered column), thedynamic range is compressed from 1:10 to ˜1:4.

Next, the region around the precursor m/z peak (±1.5 Da, the isolationrange) in the MS¹ scan and tabulated all peaks within this region wasexamined. All MS/MS scans that had a detectable MS¹ contaminant peakthat was ≧25% of the target's abundance were filtered out. The idea isthat perhaps contamination can be detected and those scans eliminatedfrom quantitative calculations. The filtered experiment reduced thenumber of quantifiable scans from 2,944 to 1,032 and only increased themeasured ratio to 1:6.0, shown in FIG. 12, panel B, filtered column.Reduction of the isolation window width can only, at best, marginallyimprove the measurements. Further, effective isolation at a resolutionbetter than 1.0 m/z is difficult. As seen in this data, the pervasiveinterference problem greatly handicaps the multiplexing potentialoffered by isobaric tags and that interfering species are often notdetectable at the MS¹ level.

McLuckey et al. pioneered the field of ion-ion chemistry and haveextensively described the use of PTR to manipulate ion charge states,purify populations, and simplify mass spectra. Here, it is proposed touse PTR to eliminate the interference associated with isobaric tagging.The idea is to use PTR to de-convolve the co-isolated contaminants fromthe precursor in m/z space, re-isolate the charge-reduced precursors,and then perform MS/MS to generate a pure population of reporter tags.Application of this approach to the mixture described above resulted ina near correct ratio of 1:8.5 with 2,360 identifications, shown in FIG.12, panel B, PTR column. As an added benefit, the reporter tag ionproduction is now easily decoupled from sequence ion generation—calledthe chaser. For instance, the scan sequence (automated by control codemodification) in FIG. 12, panel B (PTR) comprised: precursor selection(QLT), precursor purification (PTR, in QLT), isolation of thecharge-reduced product (QLT), reporter tag generation and storage (HCDcell), precursor re-injection (QLT), precursor isolation (QLT), ion trapCAD (the chaser, QLT), injection of those products into the HCD cell,and mass analysis of reporter and sequence ions together (orbitrap).This sequence was automated by control code modification. With thisapproach, alternative fragmentation methods not fully compatible withisobaric tags (i.e., ETD or ion trap CAD) can be seamlessly woven inwithout loss of quantitative information. It also affords theopportunity to optimize tag production conditions irrespective ofsequence ion generation. Finally, since +1 precursors are rarelyencountered (ESI-LC-MS), the approach is applicable to nearly alleluents.

Continued development can be achieved via duty cycle improvement,reporter signal generation, implementation of PTR reaction in the ETDcell, and PTR reagent selection. Currently the PTR/HCD scan is ˜30-40%longer than a standard HCD scan, though the implementation shows only aslight reduction in IDs from 2,944 to 2,360. This is strong performanceand, considering that interference renders the quantitative data fromthe 2,944 HCD IDs essentially useless, the PTR/HCD method is already apowerful quantification tool. Because the identification informationcomes from a separate activation event, the chaser, PTR purificationdoes not influence ID sensitivity. ID reduction can thus be attributedto duty cycle. The duty cycle can be improved by investigating PTRanions of m/z lower than the current m/z 216. Performing the PTR stepwithin the ETD cell can further increase the reaction rate, but requiressubsequent isolation of the PTR product in the QLT. Despite theresultant additional 10-15 ms and marginally decreased sensitivity(transfer losses), an overall savings of ˜30-40 ms (20% duty cycleincrease) is achieved. The proposed ETD cell does not necessarily affordisolation capability; as these modifications may in some instancesrequire considerable effort and expense. Given the method's alreadystrong performance and the proximity of the QLT, extending isolationcapability to the ETD cell is not necessary.

As shown in FIG. 12, panel A: Measurement of precursor purity typicallyobserved in an isobaric tag experiment (“w/o PTR” in the Figure) vs.purity distribution following PTR (“PTR” in the Figure). As shown inFIG. 12, panel B: Quantitative accuracy for an isobaric taggingexperiment of yeast peptides mixed in a 1:10 ratio and contaminated withhuman interference at 1:1. PTR substantially improves quantitativeaccuracy (1:8.5) and still produces comparable identifications.

The present methods are also beneficial for inducing exclusive reportertag formation. Increasing HCD energy does boost tag signal, but too muchenergy can negate any gains through scattering losses. Preliminary dataindicates that IRMPD presents an attractive alternative; by using theAI-ETD laser, primary product ions have been effectively converted toreporter tag signals (at normal QLT operating pressures). In thisexperiment, the sum of the reporter signal was just over ⅔ the intensityof the initial product signal. Automating this experiment, even withoutfurther gains, would greatly boost quantitative accuracy and dynamicrange via overall S/N improvement. Finally, the separation of reporterand sequence ion generation will enable the seamless introduction of ETDwith isobaric tagging. To implement ETD a custom control code may beused to pass the ETD reagent through the ETD cell into the QLT forion-ion reactions. This may be done because the ETD cell will be storingthe reporter ions and cannot be used for ETD. Little reaction of the +1reporter ions is anticipated for two reasons: (1) the reagents will bepassing with high relative velocity and (2) reporter ions are singlycharged and will not be particularly reactive on the time-scale of anioninjection (˜10 ms). The use of a data-dependent Decision Tree algorithmto automatically integrate ETD and CAD for shotgun proteomics has beenpreviously described. Such algorithms can used to integrate other massspectrometry methods with PTR. Standard peptide mixtures, introducedwith infusion, can be used to test reaction rates, conversionefficiencies, ETD implementation, and reporter tag generation via IRMPD.After optimization, large-scale testing by nLC-MS analysis of complexmixtures of yeast peptides can be conducted, and model interference (asdescribed above) can be conducted to evaluate the efficacy of thepurification process. Comparison of the PTR approach to the standard HCDmethod will provide duty cycle benchmarking.

The central outcome is a multiplexed quantification method with highdynamic range and accuracy. The use of PTR chemistry solves alongstanding problem with isobaric tagging. By enabling routinemultiplexing and compatibility with human tissues and biofluids, theapproach can have substantial biomedical impact.

Example 3 New Quantmode Characteristics

The QuantMode scan sequence is designed to be able to be used onETD-enabled instruments, such a LTQ Velos Orbitrap instrument. Briefly,this scan sequence comprises: precursor selection, precursor gas-phasepurification, isolation of the charge-reduced products, reporter taggeneration and storage (such as in a dedicated beam-type collisioncell), precursor re-injection, precursor isolation (QLT), ion trap CAD(QLT), injection of CAD products into the dedicated collision cell, andmass analysis of reporter and sequence ions together. This scanprogression not only produces a purified reporter tag region, but alsodecouples reporter tag ion production from sequence ion generation toenable a boost in reporter tag signal without the loss of key sequencingions. The method was validated using a mixed organism model whichmaximized the occurrence of precursor interference. Using this model,QuantMode was found to produce significant improvements in both dynamicrange and quantitative accuracy, with minimal losses in peptideidentifications.

FIG. 13A provides a plot of the measured isobaric tag ratio as afunction of detectable precursor interference in the MS isolationwindow. Even precursors having highly pure (>25%) target precursors havemeasured ratios much lower than expected (dotted line). FIG. 13Bprovides plots of measured isobaric tag ratios as a function of targetprecursor intensity. Targets of high intensity provide ratios closest tothe true value (dotted line).

FIG. 14A provides plots of measured isobaric tag ratios as a function ofreporter tag intensity. Tags of high intensity provide ratios closest tothe true value (dotted line). FIG. 14B provides plots of measuredisobaric tag ratios as a function of precursor charge state. Tags ofhigh charge states provide ratios furthest from the true value (dottedline).

FIG. 15 provides a spectrum corresponding to an isolation window showingthe isotopic cluster of the peptide precursor cation of the peptidehaving the sequence RINELTLLVQK. The spectrum shown in FIG. 15 shows thepresence of interfering species.

FIG. 16 provides a MS/MS spectrum of the precursor region shown in FIG.15. Reporter tags at m/z 126 and 127 should have ratio of 10:1. They aremeasured at 2.5:1.

FIG. 17 provides a spectrum corresponding to the reaction of theisolation window shown in FIG. 15. Multiple products are detected fromboth the target peptide and contaminating species.

FIG. 18 provides a spectrum corresponding to the isolation windowshowing a purified precursor peptide cation population following aproton transfer reaction of the isolation window shown in FIG. 15.

FIG. 19 provides a product ion spectrum following dissociation of theisolated charge-reduced target precursor population. Note the reportersignal is now measured at 9.9:1 and is almost identical to the truevalue of 10:1.

FIG. 20 provides a product ion spectrum following isolation anddissociation of the charge-reduced precursor of the peptide having thesequence TASGNIIPSSTAGAAK.

FIG. 21 provides a product ion spectrum following (1) isolation anddissociation of the charge-reduced precursor of the peptide having thesequence TASGNIIPSSTAGAAK and (2) fragmentation of the isolated noncharge-reduced precursor and combination with the products generated instep 1. Note significantly more sequence informative ions in thisspectrum.

FIG. 22 provides a schematic diagram of an algorithm illustrating thatan embodiment of the present invention (e.g., QuantMode) can beselectively applied to a sample depending on whether highly intense orhighly purified precursors are present. If a precursor ion has lowintensity at the MS¹ stage and low purity (i.e., less than 90%) thenaccurate identification and/or quantification may be unlikely even withgas-phase purification. In this instance, the algorithm may direct thisprecursor to be skipped from further mass spectrometry analysis. If theprecursor ion exhibits low intensity but has high purity, then furtheranalysis may be performed with PTR. Similarly, PTR may be utilized ifthe precursor ion exhibits high intensity but low purity. If theprecursor ion exhibits high intensity and high purity, then PTR may notbe necessary and analysis by HCD MS² may be performed.

Example 4 Low Resolution Quantmode

To extend gas-phase purification to a broader subset of proteomics, agas-phase purification platform (named “low-res QuantMode”) wasdeveloped that is amendable to standalone ion trap instruments. Thismethod also utilizes PTR to eliminate precursor interference for theimprovement of isobaric tag-based quantification and further allows thecombination of sequential HCD and CAD fragmentation into one m/zanalysis. With the development of low-res QuantMode, this functionalityhas now been extended to low resolution instruments in addition to highresolution instruments, providing a more accessible solution to theproblem of precursor interference. It is believed that such a systemwill be a valuable tool to the proteomics community, as it will enable abroader subset of researchers to confidently and effectively incorporateisobaric tag-based quantitation into their studies.

In this method, beam-type activation can be achieved using ESI ioninjection optics of stand-alone ion traps. This activation method,called front-end high energy collision-induced dissociation (fHCD), hasrecently been optimized and commercialized for use on benchtopinstruments (LTQ Velos Pro, Thermo Fisher Scientific). fHCD is ideal forisobaric tag-based quantitation on low resolution instruments, as ityields product ions which closely resemble those of HCD, it outperformsPQD in peptide identifications (2-fold improvement) and reporter ionintensities (10-fold improvement), and, unlike CAD, it allows for theretention of reporter ions.

The scan sequence further comprises three separate scan events: 1)charge-state determination, 2) quantification, and 3) identification.Incorporation of these specialized scan events and fHCD into theQuantMode scan sequence enabled the development of a low-resolutiongas-phase purification method which improves quantitative accuracy,maximizes peptide identifications, and minimizes unnecessary drains onduty cycle. Herein, the low-res QuantMode scan function is described indetail along with improvements provided by this method for isobarictag-based quantification through the analysis of both a mixed organismmodel (which replicates a worst-case precursor interference scenario)and a complex biological time course sample.

Cell Culture and Differentiation

Wild-type yeast (Saccharomyces cerevisiae) was grown in rich medium toan OD600 of 0.6. Cells were collected and centrifuged at 14,200 g for 10min at 4° C. The resulting cell pellet was washed twice with sterilewater and centrifuged at 1,100 g for 5 min. Lysis buffer ofapproximately three times the cell pellet volume was added. The lysisbuffer contained 8 M urea, 75 mM NaCl, 50 mM Tris (pH 8), 10 mM sodiumpyrophosphate, 100 mM sodium butyrate, complete mini ETDA-free proteaseinhibitor (Roche Diagnostics) and phosSTOP phosphatase inhibitor (RocheDiagnostics). The cells were lysed using a French press, where thesample was pressed three times and then centrifuged for 15 min at 24,000g at 4° C.

Human H1 embryonic stem cells were maintained in a feeder-independentsystem [Ludwig et al., Nat. Materials, 2006, 3, p. 637-646]. Cells werecollected by application of an adequate volume of prewarmed (37° C.)0.05% Tryp-LE to cover the culture surface for 10 min. After celldetachment, an equivalent volume of ice-cold DPBS (Invitrogen) was addedbefore collecting the cells. Cell pellets were subsequently washed twicein ice-cold PBS and stored at −80° C. Cells were lysed via sonication inlysis buffer containing 30 mM NaCl, 50 mM Tris (pH 8), 2 mM MgCl₂, 50 mMNaF, 1 mM sodium orthovanadate, 6 mM sodium pyrophosphate, miniEDTA-free protease inhibitor (Roche Diagnostics) and phosSTOPphosphatase inhibitor (Roche Diagnostics).

C2C12 mouse myoblasts were cultured as subconfluent monolayers in DMEMhigh glucose medium (Invitrogen) with 10% fetal bovine serum(Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin(Invitrogen) and incubated at 37° C. and 5% CO₂. To induce myotubedifferentiation, myoblasts were grown to confluence then switched tomedia containing 2% horse serum (Invitrogen). The cells were re-fedevery 24 hours during the differentiation. Undifferentiated myoblastswere collected the day of the media change (day 0) and fullydifferentiated myotubes were collected 6 days following the media change(day 6). Cells were collected by dissociation with 0.05% Trypsin-EDTA(Invitrogen), washed with PBS, pelleted by centrifugation, snap frozenin liquid nitrogen and stored at −80° C. until use.

Cell pellets were resuspended in approximately three times the pelletvolumes using lysis buffer. The lysis buffer contained 8 M urea, 50 mMTris (pH 8), 40 mM NaCl, 2 mM MgCl₂, 50 mM NaF, 50 mM b-glyceraldehydephosphate, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, miniEDTA-free protease inhibitor (Roche Diagnostics), and phosSTOPphosphatase inhibitor (Roche Diagnostics). Cells were lysed viasonification. Protein content within each of the mouse samples wasevaluated using a BCA assay (Thermo).

Cell Lysis and Digestion

Yeast, human, and mouse proteins were subject to cysteine residuereduction using 5 mM DTT and alkylation using 10 mM iodoacetamide.Proteins were then digested using a two-step process. First, ProteinaseLys-C (Wako Chemicals) was added to each sample at a ratio of 1:100(enzyme:protein) and the resulting mixtures were incubated at 37° C. for4 hours. Next, samples were diluted to a final concentration of 1.5 Murea (pH 8) with a solution of 25 mM tris and 2 mM CaCl.Sequencing-grade trypsin (Promega) was added to each sample at a ratioof 1:100 (enzyme:protein) and the resulting mixtures were incubated at37° C. overnight. Reactions were quenched using trifluoroacetic acid.Samples were desalted using C18 solid-phase extraction columns (SepPak;Waters) and dried to completion.

Sample Preparation

Interference Sample:

Yeast peptides were split into six equal mass aliquots; each aliquot waslabeled with one of six TMT 6-plex reagents (m/z 126-131), as describedpreviously (TMTsource). Yeast aliquots were mixed in the mass ratios1:5:10:10:5:1, respectively. Human peptides were split into three equalmass aliquots; each aliquot was labeled with one of three TMT 6-plexreagents (m/z 129-131), as described previously (TMTsource). Humanaliquots were mixed in the mass ratios 1:1:1, respectively. A smallaliquot was obtained from each individual sample to provide material forcontrol experiments. Yeast and human samples were then combined in a 2:1mass ratio, respectively. All samples were dried to completion andresuspended in 0.2% formic acid for LC-MS analysis.

Myoblast Differentiation Sample.

The ‘day 0’ and ‘day 6’ mouse samples were split into three equal massaliquots; each of the ‘day 0’ aliquots was labeled with one of thelightest three TMT 6-plex reagents (m/z 126-128) while each of the ‘day6’ aliquots was labeled with one of the heaviest three TMT 6-plexreagents (m/z 129-131), as described previously (TMTsource). The sixresulting aliquots were desalted using C18 solid-phase extractioncolumns, dried to completion, and combined in equal masses. The labeledmouse peptide mixture was fractionated using SCX. Eight fractions werecollected over the SCX gradient: two 6-min intervals, five 1-minintervals, and one 10-min interval. Each fraction was lyophilized,desalted, dried to completion, and resuspended in 0.2% formic acid forLC-MS analysis.

Liquid Chromatography-Mass Spectrometry

All experiments were performed using a NanoAcquity UPLC system (Waters)coupled to an ETD-enabled LTQ Velos mass spectrometer (Thermo). AnanoESI source was used for the generation of precursor peptide cations.Samples were loaded onto a precolumn (75 μm i.d., packed with 10 cm of 5μm C18 particles; Microm Bioresources, Inc) for 15 minutes at 98:2 0.2%formic acid:acetonitrile with 0.2% formic acid at a flow rate of 0.850μL/min. Samples were then separated on an analytical column (75 μm i.d.,packed with 15 cm of 5 μm C18 particles; Microm Bioresources, Inc) at aflow rate of 0.300 μL/min using a gradient consisting of an initialsteep rise to 7% B (acetonitrile with 0.2% formic acid) followed by a 90minute linear gradient from 7% to 30% B and a final ramp to 70% B over 4minutes which was held for 5 minutes. The column was equilibrated with2% buffer B for an additional 20 minutes. Precursor peptide cations weregenerated from the eluent through the utilization of a nanoESI source.

All non-QuantMode instrument methods consisted of an MS¹ scan (300-1600m/z) followed by ten data-dependent trapHCD MS² scans, all analyzed inthe ion trap at a normal scan speed. MS² scans employed a precursorisolation window of 3 Th and a trapHCD normalized collision energy (NCE)setting of 60 for 2 ms.

All QuantMode instrument methods consisted of an MS¹ scan (300-1600 m/z)followed by three data-dependent QuantMode scan cycles, all analyzed inthe ion trap. Precursor isolation windows of 3 Th were used. TheQuantMode scan cycle utilizes proton transfer reactions (PTR) to achievegas-phase purification. For all experiments, the nitrogen adduct offluoranthene (m/z 216) was used as the PTR reagent ion. Reagent anionswere generated by an integrated chemical ionization source (commercialETD module; Thermo); source conditions and all associated ion opticswere optimized for this reagent prior to each set of experiments.

The QuantMode scan cycle was composed of three separate scan events: 1)charge-state determination scan; 2) quantitation scan(s); and 3)identification scan. The charge-state determination scan was an MS²event in which isolated precursor cations were subjected toresonant-excitation CAD (q-value=0.25, NCE 1, 1 ms) and analyzed in theion trap at an enhanced scan speed over a 15 Th region surrounding theprecursor isolation window. Instrument code was modified to store thecharge-state of each isolated precursor population for the subsequentscan (charge-state prediction based on the m/z of ions surrounding theMS² base peak). The quantitation scan was an MS³ event. Isolatedprecursor cations and reagent anions were sequestered in the highpressure trap and comingled via charge-sign independent trapping. Theduration of the PTR reaction was dictated by the charge-state predictionin the previous scan event to ensure optimal reaction conditions forsingle charge reduction. Reaction times were set at 80 ms for doublycharged precursors, 30 ms for triply charged precursors, and 20 ms forquadruply charged precursors. Following PTR, the charge-reducedprecursor population was re-isolated based on the predictedcharge-state. If no charge-state was predicted, the precursor wasre-isolated as if it were initially a triply charged cation. There-isolated precursors then underwent trapHCD activation [NCE 90(optimal for tag generation), 2 ms] followed by ion trap analysisscanned at normal scan speed over a 30 Th window surrounding the TMT-tagregion (110-140 m/z). If no charge-state was determined for theprecursor of interest, a second quantitation scan was performed in whichprecursors were treated as quadruply charged cations. Instrument codewas modified to skip this second quantitation scan if charge-state wasdetermined. The identification scan was an MS² event in which isolatedprecursor cations were subjected to trapHCD [NCE 70 (for doubly-chargedprecursors)/NCE 50 (for all other precursors), 2 ms] and analyzed in theion trap at a normal scan speed over the full mass range.

The automatic gain control (AGC) target settings for precursor cationswere 4×10⁴ for MS¹ scans, 1×10⁴ for CAD-activated MS² scans, 4×10⁴ fortrapHCD-activated MS² scans, and 1.2×10⁵ for PTR quantitation scans.Precursors were subject to dynamic exclusion for 60 seconds using awindow of −0.5 to 2.5 Th. The reagent anion AGC target setting was2×10⁵.

Data Analysis

Data was processed using the in-house software suite COMPASS. OMSSA(version 2.1.8) searches for interference sample data were performedagainst the International Protein Index (IPI:www<dot>ebi<dot>ac<dot>uk/IPI/) target-decoy database comprised of bothyeast (Saccharomyces Genome Database, www<dot>yeastgenome<dot>org, “all”version including all systematically named open reading frames (ORFs),including verified, uncharacterized, and dubious ORFs and pseudogenes)and human (version 3.80) proteins. OMSSA searches for myoblastdifferentiation data were performed against the Universal ProteinResource (UNIPROT: www<dot>uniprot<dot>org/) target-decoy mousedatabase. Searches were conducted using an average precursor masstolerance of ±5.0 Da and a monoisopic product mass tolerance of ±0.50Da. The fixed modifications specified were carbamidomethylation ofcysteines, TMT 6-plex on peptide N-termini, and TMT 6-plex on lysines.The variable modifications specified were oxidation of methionine andTMT 6-plex on tyrosines. A maximum of 3 missed tryptic cleavages wereallowed. Interference data was independently trimmed to 1% FDR andsubsequently filtered to remove all human-derived peptides, enablingonly yeast-derived peptides to be considered for analysis. Myoblastdifferentiation data fractions were collectively filtered to 1% FDR foreach set of experiments. Noise-band capping of missing TMT 6-plexchannels was manually applied during quantitative analyses based on thelowest TMT peaks detected within each set of experiments. Statisticaland GO/KEGG-term analyses of myoblast differentiation data wereconducted using the Persius software package [Cox, J. and Mann, M., NatBiotechnol, 2008, 26, pgs. 1367-72].

Low-Res QuantMode Scan Sequence

Instrument software was modified to enable implementation oflow-resolution QuantMode (low-res QuantMode) on a stand-alone ion trap.A full low-res QuantMode scan sequence subjects a precursor to threeseparate scan events: a charge-state determination scan, one or morequantitation scans, and an identification scan. This workflow isoutlined in FIG. 23, and each scan event within the cycle is presentlydescribed.

Charge-State Determination Scan.

Unlike orbitrap mass analyzers, ion trap mass analyzers cannot providethe spectral resolution necessary to elucidate charge-state informationfrom sampled precursor ions. Knowledge of precursor charge-state iscritical for the implementation of PTR within the low-res QuantModemethod; without this information, re-isolation of the charge-reducedprecursor is inefficient, at best. For this reason, the first low-resQuantMode scan event was dedicated entirely to the acquisition ofprecursor charge-state.

Charge-state determination was achieved in this first scan event byisolating precursor ions in a 3Th m/z window and performing m/z analysison the isolated region using a slower, ‘enhanced’ scan rate. Thisenhanced scan rate improved baseline resolution to enable subsequentcharge state determination for doubly and triply protonated precursorpeptides.

The utility of the targeted enhanced scan for charge state predictionwas evaluated outside of the low-res QuantMode method using a sample oftryptic, unlabeled mouse peptides. A 90 minute data-dependent top 3method subjected each precursor to three scan events: 1) the low-resQuantMode charge-state determination scan; 2) PTR followed by full m/zanalysis; and 3) CID (35 NCE) followed by full m/z analysis. The lattertwo scans in this method provide charge-state verification for thetargeted scan. A computer algorithm was written which determinedprecursor charge-state from PTR spectra based on the intensity and m/zlocations of the charge-reduced precursors. OMSSA identificationsobtained from CID spectra provided another source of charge-stateinformation for each sampled precursor.

These verification scans allowed confirmation that a targeted scanprediction of a doubly or triply charged precursor was correct at least88% of the time (FIG. 24 a). Despite this high success rate, however,the targeted scan was only able to predict charge state information for42% of all precursors sampled, and only 60% of these predictedcharge-states were associated with peptides amenable to PTR (≧+2 charge)(FIG. 24 b). Although the targeted scan correctly predicted charge-stateinformation for over half of the doubly charged precursors identified inthe evaluation experiment, it generated significantly less predictionsfor the triply charged—and no predictions for the quadruplycharged—precursors (FIG. 24 c). Peptides labeled with isobaric tags tendto ionize to higher charge states than non-labeled peptides, makinghighly-charged precursors with un-identified charge states too importantto completely ignore (FIG. 25). The charge state determination scan wastherefore mainly utilized as a screening process; if no charge state wasdetermined, the peptide was evaluated as both a triply and quadruplycharged peptide for quantitation purposes. Charge-state was laterconfirmed during data processing using PSM information gleaned from theidentification scan. By eliminating the need to quantitatively evaluateeach peptide as doubly, triply, and quadruply charged species,integration of the targeted scan into low-res QuantMode actually enableda shorter duty cycle and a greater amount of peptide identifications(FIG. 24 d). It should be noted that performing this targeted scan usinga slower ‘zoom’ scan speed resulted in no improvement in charge-stateidentification and negatively affected both duty cycle and total peptideidentifications (data not shown).

Quantitation Scan(s).

Peptide quantification was performed in the second scan event throughthe utilization of gas-phase purification. Precursor ions were subjectedto PTR and re-isolated based on the charge-state determined in theprevious scan. This ‘purified’ charge-reduced precursor population wasthen fragmented with fHCD activation at a collision energy optimal fortag generation. As was mentioned above, in cases where charge-stateinformation was unavailable, two quantitation scans were performed: onewhich assumed a precursor charge state of +3 and one which assumed aprecursor charge state of +4. Whether performing a single or multiplequantitation scans, m/z analysis was only performed for a narrow rangecorresponding to the reporter tag m/z region; in so doing, the dutycycle penalty associated with multiple MS/MS events was drasticallyreduced.

Identification Scan.

Lastly, an identification scan was performed to ascertain each peptidesequence. Useful sequence ions were generated using fHCD activation.Alternatively, this identification scan could have been performed usingCAD activation; however, it was found that trapHCD activation produced agreater number of peptide spectral matches (data not shown).

Segmenting the low-res QuantMode scan sequence into three distinctevents enabled the decoupling of sequence and reporter ion generation;this increased the likelihood of obtaining accurate quantitation andsequence information from each peptide analyzed. Optimal activationconditions for peptide identification and tag generation were determinedexperimentally (FIG. 26). The optimal collision energy for high reporterion signal intensity was independent of charge-state, which enabled asingle collision energy (NCE 90) to be used for all quantitation scans(FIG. 26). The optimal collision energy for the generation of usefulsequence ions, however, was dependent on charge-state (FIG. 26). Sincecharge-state information isn't typically available on low-resolutioninstruments, methods scale activation energies based on a default chargestate (set to +3 in all of these experiments); all peptides aretherefore subject to the same collision energies, regardless of theiractual charge-state. In the low-res QuantMode analyses, it was possibleto take advantage of the charge-state determination scan to scale up theactivation energy for +2 precursors and scale down the activation energyfor all other precursors. Overall, this minimized the incidence ofunderreacted/overreacted spectra and maximized peptide identificationsin the experiments.

Evaluation Of Low-Res Quantmode Using Interference Model

To evaluate the ability of low-res QuantMode to remedy the problem ofprecursor co-isolation in isobaric tag-based quantification, the methodwas first tested using a mixed organism model designed to mimic a‘worst-case’ scenario for precursor interference. To generate this mixedorganism sample, yeast peptides labeled with TMT tags 126, 127, 128,129, 130, or 131 were mixed in a 1:5:10:10:5:1 ratio, respectively. Theyeast sample was then contaminated with an equivalent amount of humanpeptides carrying the tags 129, 130, and 131 (mixed in a 1:1:1 ratio,respectively). LC-MS/MS analysis of the interference sample highlightsthe breakdown of quantitative accuracy which occurs in the reporter tagregion when multiple species are co-isolated; any yeast-identifiedpeptide containing human interference will contain skewed 5:1 and 10:1ratios in the right-most channels while maintaining the correct 5:1 and10:1 ratios in the left-most channels. For the purpose of this analysis,the left-most channels (126-128) will be referred to as ‘controlchannels’; control channel ratios are determined by comparing tags127:126 and 128:126 (5:1 and 10:1 ratios, respectively). Similarly, theright-most channels (129-131) will be referred to as ‘interferencechannels’; interference channel ratios are determined by comparing tags130:131 and 129:131 (5:1 and 10:1 ratios, respectively).

Baseline quantitation was established through fHCD analysis(data-dependent top 10 (ddTop10), NCE 60) of the yeast control (i.e.,the interference sample prior to the addition of human peptides). Withno interfering species present, 4.8:1 and 8.9:1 ratios in the controlchannels and 4.5:1 and 8.2:1 ratios in the interference channels wereobserved. Deviation from the expected 5:1 and 10:1 ratios can beattributed to sample preparation inconsistencies.

The interference sample (composed of both yeast and human peptides inthe amounts specified above) was analyzed twice, once using only fHCD(ddTop10, NCE60) and once using QuantMode (as described). Data wasfiltered to provide quantitative results for only the yeast peptidesidentified in each experiment; outcomes are shown in FIG. 27. Theseverity of the precursor interference problem is demonstrated by thetruncated ratios observed in the fHCD-only analysis. While controlchannels retained ratios of 4.9:1 and 9.2:1, interference channelsshowed incredibly truncated ratios of 1.8:1 and 2.6:1. This translatesto a 3-fold underestimation of the 129:131 ratio and a 2.5-foldunderestimation of the 130:131 ratio. Implementation of low-resQuantMode, however, was able to recover these diminished proportions.Again, control channels retained ratios of 4.9:1 and 9.2:1, butinterference channels now displayed ratios of 3.0:1 and 4.9:1—numberswhich are significantly closer to the respective 4.1:1 and 8.2:1 ratiosobserved in the yeast control. This marks a 28% and 27% improvement inquantitative accuracy for the 129:131 and 130:131 reporter tag ratios(32% to 60% and 40% to 67%), respectively, when comparing quantitativeanalyses conducted with and without low-res QuantMode.

The low-res QuantMode scan cycle provides dramatic improvements inquantitative accuracy, but does so at the slight expense of peptide andprotein identifications. Despite efforts to make the method as efficientas possible, the low-res QuantMode duty cycle is significantly slowerthan the fHCD-only duty cycle, which translates to a 53% loss in peptideidentifications and a 37% loss in protein identifications. This begs thequestion of whether it is more beneficial to sacrifice quantitativeaccuracy or high sequence coverage when analyzing complex samples.Quantitative analysis of a real, complex biological time course sampleadvocates for the former, and the results of this study are presentlyrevealed.

Evaluation of Low-Res QuantMode using C2C12 Myogenesis Sample

Interference model experiments establish improvements in quantitativeaccuracy when using low-res QuantMode in a ‘worst case’ interferencescenario; however, it is demonstrated that the utility of low-resQuantMode translates to the analysis of large-scale, complex biologicalsamples as well.

The differentiation of mouse-derived C2C12 myoblasts has beenextensively studied over the past decade as a model system for thedevelopment and interaction of skeletal muscle myocytes. Over the courseof six days, C2C12 myoblasts undergo myogenic differentiation to formmyotubes, and this development process is accompanied by dynamic changesin protein expression. In recent years, quantitative mass spectrometrymethods, such as spectral counting and SILAC, have been utilized toinvestigate these myogenic protein dynamics. All studies findsignificant changes in the presence of metabolic and structural proteinsduring various stages of the differentiation process.

To validate the quantification advantages provided by the low-resQuantMode method, both fHCD (ddTop10, NCE60) and low-res QuantMode wereused to compare relative protein levels present in the myogenic cells atday 0 and day 6 of the differentiation process. Myoblast (day 0) andmyotube (day 6) cells were separately harvested, lysed, digested andsplit into three equal mass aliquots. Each myoblast aliquot wasseparately labeled with TMT 6-plex tags 126 to 128 m/z while eachmyotube aliquot was separately labeled with TMT 6-plex tags 129-131 m/z.All aliquots were combined in equal mass ratios; the resulting samplewas fractionated with SCX and analyzed using both fHCD and QuantModeLC-MS methods.

The results of this analysis are presented in FIG. 28. As was observedin the interference experiments, a faster duty cycle enables fHCD-onlyanalyses to identify a greater overall number of proteins than low-resQuantMode analyses (4050 vs. 2964, respectively). When assessing thenumber of protein identifications associated with changes greater than1.5-fold, however, the two methods become comparable; in fact, low-resQuantMode actually identified more 1.5-fold changes than fHCD by amargin of almost 15% (1326 vs. 1132, respectively). Improvements indynamic range only become more substantial as low-res QuantMode and fHCDidentifications are compared at higher fold-changes; low-res QuantModeproduced significantly more >2-fold (766 vs. 438) and >5-fold (96 vs.12) changes overall (FIG. 28 a). Given the 1332 proteins detected inboth sets of experiments, greater fold-changes were discovered, onaverage, when proteins were analyzed using low-res QuantMode (FIGS. 28 band 28 c). In a large-scale time course study such as this one, soundquantitative accuracy and high dynamic range are essential for thedetermination of biological relevance.

FIG. 29 illustrates this point by presenting typical MS² data obtainedfor two proteins discovered during the course of both experiments. Thelevels of both proteins significantly change over the six day timecourse; however, the differences in protein levels are significantlymore pronounced in QuantMode analysis. This suggests that these proteinsmay change more dynamically in the differentiation process than wouldhave been thought given only fHCD data. By purifying the precursorpopulation prior to fragmentation, the QuantMode scan function generatesdata that is significantly more accurate, and therefore more useful, forbiological applications.

Although the description herein contains many specifics, these shouldnot be construed as limiting the scope of the invention, but as merelyproviding illustrations of some of the embodiments of the invention.

Each reference cited herein is hereby incorporated by reference in itsentirety. However, if any inconsistency arises between a cited referenceand the present disclosure, the present disclosure takes precedent. Somereferences provided herein are incorporated by reference to providedetails concerning the state of the art prior to the filing of thisapplication, other references can be cited to provide additional oralternative device elements, additional or alternative materials,additional or alternative methods of analysis or applications of theinvention. Patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although theinvention has been specifically disclosed by preferred embodiments,exemplary embodiments and optional features, modification and variationof the concepts herein disclosed can be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.The specific embodiments provided herein are examples of usefulembodiments of the invention and it will be apparent to one skilled inthe art that the invention can be carried out using a large number ofvariations of the devices, device components, methods steps set forth inthe present description. As will be obvious to one of skill in the art,methods and devices useful for the present methods can include a largenumber of optional composition and processing elements and steps.

One of ordinary skill in the art will appreciate that device elements,as well as materials, shapes and dimensions of device elements, as wellas methods other than those specifically exemplified can be employed inthe practice of the invention without resort to undue experimentation.All art-known functional equivalents, of any such materials and methodsare intended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedcan be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and possible subcombinationsof the group are intended to be individually included in the disclosure.Every combination of components or materials described or exemplifiedherein can be used to practice the invention, unless otherwise stated.One of ordinary skill in the art will appreciate that methods, deviceelements, and materials other than those specifically exemplified can beemployed in the practice of the invention without resort to undueexperimentation. All art-known functional equivalents, of any suchmethods, device elements, and materials are intended to be included inthis invention. Whenever a range is given in the specification, forexample, a temperature range, a frequency range, a time range, or acomposition range, all intermediate ranges and all subranges, as wellas, all individual values included in the ranges given are intended tobe included in the disclosure. Any one or more individual members of arange or group disclosed herein can be excluded from a claim of thisinvention. The invention illustratively described herein suitably can bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. The term “comprising”is intended to be broader than the terms “consisting essentially of” and“consisting of”, however, the term “comprising” as used herein in itsbroadest sense is intended to encompass the narrower terms “consistingessentially of” and “consisting of”, thus the term “comprising” can bereplaced with “consisting essentially of” to exclude steps that do notmaterially affect the basic and novel characteristics of the claims and“comprising” can be replaced with “consisting of” to exclude not recitedclaim elements.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed can be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Although the description herein contains many specifics, these shouldnot be construed as limiting the scope of the invention, but as merelyproviding illustrations of some of the embodiments of the invention.

What is claimed is:
 1. A method of analyzing an analyte using massspectrometry, the method comprising: (a) providing an isobaricallylabeled analyte; (b) generating a first distribution of precursor ionsfrom the isobarically labeled analyte; (c) identifying a range ofmass-to-charge ratios of the first distribution of precursor ions; (d)selectively changing the mass-to-charge ratios of precursor ions in theidentified range of mass-to-charge ratios, thereby generating adistribution of mass-to-charge-manipulated precursor ions; (e) isolatinga range of mass-to-charge ratios of the distribution ofmass-to-charge-manipulated precursor ions, thereby generating isolatedmass-to-charge-manipulated precursor ions; (f) fragmenting ionscorresponding to the isolated mass-to-charge-manipulated precursor ions,thereby generating first product ions; and (g) measuring themass-to-charge ratios of the first product ions, thereby generatingfirst product ion mass spectrometry data; thereby analyzing the analyteusing mass spectrometry.
 2. The method of claim 1, further comprising:(h) generating a second distribution of precursor ions from theisobarically labeled analyte; (i) identifying a range of mass-to-chargeratios of the second distribution of precursor ions; (j) fragmentingions corresponding to the range of mass-to-charge ratios of the seconddistribution of precursor ions, thereby generating second product ions;and (k) measuring the mass-to-charge ratios of the second product ions,thereby generating second product ion mass spectrometry data.
 3. Themethod of any of claim 1, wherein the steps of: (g) measuring themass-to-charge ratios of the first product ions, thereby generatingproduct ion mass spectrometry data; and (k) measuring the mass-to-chargeratios of the second product ions, thereby generating product ion massspectrometry data; are performed sequentially or concurrently using asingle mass analyzer.
 4. The method of claim 1, wherein the firstdistribution of precursor ions is subject to collisional dissociation orphotodissociation to selectively change the mass-to-charge ratios ofprecursor ions in the identified range of mass-to-charge ratios, therebygenerating a distribution of mass-to-charge-manipulated precursor ions.5. The method of claim 1, wherein the first distribution of precursorions is subject to a reaction with a species to selectively change themass-to-charge ratios of precursor ions in the identified range ofmass-to-charge ratios, thereby generating a distribution ofmass-to-charge-manipulated precursor ions, wherein the reaction with aspecies to selectively change the mass-to-charge ratios of the range ofmass-to-charge ratios comprises a proton-transfer reaction, acharge-transfer reaction, or an electron-transfer reaction.
 6. Themethod of claim 5 wherein the proton transfer reaction comprisesreaction of the first distribution of precursor ions with fluoranthene,perfluoro-1,3-dimethyl-cyclohexane, 2,6-dichlorophenol,2,3,4,6-tetrachlorophenol, or 1,4-naphthoquinone.
 7. The method of claim1, wherein the identified range of mass-to-charge ratios of the firstdistribution of precursor ions in step (c) has a width of 3 m/z units orless.
 8. The method of claim 1, wherein the step of: (e) isolating arange of mass-to-charge ratios of the distribution ofmass-to-charge-manipulated precursor ions, thereby generating isolatedmass-to-charge-manipulated precursor ions; comprises isolating a rangeof mass-to-charge ratios of the distribution ofmass-to-charge-manipulated precursor ions corresponding to 3 m/z unitsor less.
 9. The method of claim 1, wherein the precursor ions in theidentified range of mass-to-charge ratios have a positive charge state,and selectively changing the mass-to-charge ratios of the precursor ionsin the identified range comprises decreasing the positive charge stateof each of the precursor ions by 1, by 2, or by
 3. 10. The method ofclaim 1, wherein at least a portion of the first product ion massspectrometry data corresponds to a reporter tag of the isobaricallylabeled analyte.
 11. The method of claim 1, wherein the isobaricallylabeled analyte comprises proteins or peptides and the isobaricallylabeled analyte is analyzed to quantify the amount of proteins orpeptides in the analyte.
 12. A mass spectrometer system for analyzing anisobarically labeled analyte, the system comprising: an ion source forgenerating ions from the isobarically labeled analyte; first ionseparation optics in communication with the ion source for separatingions according to their mass-to-charge ratios; ion reaction optics incommunication with the first ion separation optics for generatingmass-to-charge-manipulated ions; ion fragmentation optics incommunication with the first ion separation optics for generatingproduct ions; second ion separation optics in communication with the ionfragmentation optics for separating ions according to theirmass-to-charge ratios; a first ion detector in communication with thesecond ion separation optics for detecting ions separated according totheir mass-to-charge ratios; a controller operably connected to thefirst and second ion separation optics, the ion reaction optics, thefirst ion detector, and the ion fragmentation optics; wherein thecontroller controls the ion optics and detector so as to: (a) generate afirst distribution of precursor ions from the isobarically labeledanalyte; (b) identify a range of mass-to-charge ratios of the firstdistribution of precursor ions; (c) selectively change themass-to-charge ratios of precursor ions in the identified range ofmass-to-charge ratios, thereby generating a distribution ofmass-to-charge-manipulated precursor ions; (d) isolate a range ofmass-to-charge ratios of the distribution of mass-to-charge-manipulatedprecursor ions, thereby generating isolated mass-to-charge-manipulatedprecursor ions; (e) fragment ions corresponding to the isolatedmass-to-charge-manipulated precursor ions, thereby generating firstproduct ions; (f) measure the mass-to-charge ratios of the first productions, thereby generating first product ion mass spectrometry data; and(g) analyze the first product ion mass spectrometry data.
 13. The systemof claim 12, wherein the controller further controls the ion optics anddetector so as to: (h) generate a second distribution of precursor ionsfrom the isobarically labeled analyte; (i) identify a range ofmass-to-charge ratios of the second distribution of precursor ions; (j)fragment ions corresponding to the range of mass-to-charge ratios of thesecond distribution of precursor ions, thereby generating second productions; (k) measure the mass-to-charge ratios of the second product ions,thereby generating second product ion mass spectrometry data; and (l)analyze the second product ion mass spectrometry data.
 14. The system ofclaim 12, wherein the controller further controls the ion optics anddetectors to: subject the first distribution of precursor ions toreaction with a species to selectively change the mass-to-charge ratiosof precursor ions in the identified range of mass-to-charge ratios,thereby generating a distribution of mass-to-charge-manipulatedprecursor ions, wherein the reaction with a species to selectivelychange the mass-to-charge ratios of the range of mass-to-charge ratioscomprises a proton-transfer reaction.
 15. The system of claim 12,further comprising a second ion detector in communication with the firstion separation optics for detecting ions separated according to theirmass-to-charge ratios and generating first precursor ion massspectrometry data corresponding to the first distribution of precursorions.
 16. The system of claim 12, wherein the controller controls theion optics and detector so as to: (e) fragment ions corresponding to theisolated mass-to-charge-manipulated precursor ions, thereby generatingfirst product ions; wherein the isolated mass-to-charge-manipulatedprecursor ions are fragmented by beam-type collisionally activateddissociation, ultraviolet photo-dissociation, infraredphotodissociation, electron transfer dissociation, electron capturedissociation, surface induced dissociation, or resonant excitationcollisionally activated dissociation.
 17. The system of claim 12,wherein the isobarically labeled analyte comprises proteins or peptidesand the controller controls the ion optics and detectors to analyze theisobarically labeled analyte to quantify the amount of proteins orpeptides in the analyte.
 18. The method of claim 12, wherein theidentified range of mass-to-charge ratios of the second distribution ofprecursor ions does not include ions having a mass-to-charge ratio lessthan 200 m/z units.
 19. A method of analyzing an analyte using massspectrometry, the method comprising: (a) providing an isobaricallylabeled analyte; (b) generating a first distribution of precursor ionsfrom the isobarically labeled analyte; (c) identifying a range ofmass-to-charge ratios of the first distribution of precursor ions; (d)scanning the precursor ions in the identified range of mass-to-chargeratios to determine the charge state of the identified precursor ions;(e) reacting the precursor ions in the identified range ofmass-to-charge ratios with a species to selectively change themass-to-charge ratios of precursor ions in the identified range ofmass-to-charge ratios, wherein the reaction with a species toselectively change the mass-to-charge ratios of the range ofmass-to-charge ratios comprises a proton-transfer reaction therebygenerating a distribution of mass-to-charge-manipulated precursor ionshaving a decreased positive charge state; (f) isolating a range ofmass-to-charge ratios of the distribution of mass-to-charge-manipulatedprecursor ions, thereby generating isolated mass-to-charge-manipulatedprecursor ions; (g) fragmenting ions corresponding to the isolatedmass-to-charge-manipulated precursor ions, thereby generating firstproduct ions; and (h) measuring the mass-to-charge ratios of the firstproduct ions, thereby generating first product ion mass spectrometrydata, thereby analyzing the analyte using mass spectrometry.
 20. Themethod of claim 19, wherein if the determined charge state of theidentified precursor ions is +2 or greater, then isolating the range ofmass-to-charge ratios of the distribution of mass-to-charge-manipulatedprecursor ions is based on the determined charge state of a desiredprecursor ion.
 21. The method of claim 19, wherein if the determinedcharge state of the identified precursor ions is unknown or less than+2, then isolating the range of mass-to-charge ratios of thedistribution of mass-to-charge-manipulated precursor ions is based on adesired charge state of +3.
 22. The method of claim 19, furthercomprising: (i) isolating a second range of mass-to-charge ratios of thedistribution of mass-to-charge-manipulated precursor ions, therebygenerating second isolated mass-to-charge-manipulated precursor ions;(j) fragmenting ions corresponding to the second isolatedmass-to-charge-manipulated precursor ions, thereby generating secondproduct ions; and (k) measuring the mass-to-charge ratios of the secondproduct ions, thereby generating second product ion mass spectrometrydata.